NZ717409A - Synergistic liposomal formulation for the treatment of cancer - Google Patents

Abstract

The present disclosure relates to the synergistic liposomal formulation comprising, phophatidylcholine, stearylamine and anticancer drugs for the treatment of cancer. The PC:SA cationic liposome encapsulated camptothecin (CPT) and doxorubicin (DOX) formulations show enhanced synergistic anti-cancer effect and provide improved therapeutic index as compared to either the liposome or drug alone. The present disclosure also relates to the use of Cationic liposomal preparation of phosphatidylcholine:stearylamine (PC:SA) showing anticancer effect. The SA-bearing liposome and drug entrapped in the liposome are effective against cancer both in vitro and in vivo, without causing any adverse effect on host.

Description

SYNERGISTIC LIPOSOMAL FORMULATION FOR THE TREATMENT OF CANCER FIELD OF THE INVENTION [0001} The present disclosure relates to a synergistic mal formulation. The stearylamine bearing cationic liposome is shown to be very effective against the treatment of the disease cancer, and when anticancer drug camptothecin is ulated into the cationic liposome, then the formulation shows anticancer property which is far more effective than either the liposome or the drug alone.

' OUND OF THE INVENTION : [0002] A major lty of targeted drug delivery against cancer is the lack of ubiquitously sed tumour¥specific antigen or receptor. However, it has now been established that the- phospholipid, Phosphatidylserine (PS) can be exploited as a ial target for drug delivery against cancer. id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3" id="p-3"

[0003] PS is a phospholipid that under normal conditions resides almost exclusively in the inner leaflet of the'plasma membrane. PS asymmetry is maintained by an ATP- dependent amino phospholipid translocase that is responsible for inward nt of the aminophospholipids. Loss of the PS asymmetry is observed under different ogic and physiologic conditions, including programmed cell death, cell aging, cell migration, cell degranulation (l). Spontaneous PS exposure has been observed in many malignant cell types in the absence of exogenous activators or cell injury (2).

Surface PS exposure is observed .in solid tumors and tumor vasculature and can be a marker oftumor vasculature (3). ressing tumor endothelium was mostly found to [be non-apoptotic. The factors like hypoxic reoxygenation, inflammatory cytokines, ‘ acidity, all mostly prevalent factors in tumorigenesis, are mainly the cause of PS exposure. Recent s have shown that tumorigenic, undifferentiated murine erythroleukemia cell expresses 7—8 fold more PS in their outer leaflet than their differentiated counterparts (2). Elevated expression of surface PS is also found in human melanoma and lung carcinoma (4). Hence, PS on tumor vessels is an attractive target for cancer imaging and therapy. Reports have shown that anti-cancer drugs have been prepared exploiting the elevated PS level of the cancer cells which serves as a U1 marker. NK-l‘ysin derived peptide NK-2 preferentially kills cancer cells. The selectivity ofthe cationic membranolytic peptide NK-2 has been assigned to the differences inthe membrane phospholipid ition of target cancer cells (5). PS exposure isneither of apoptotic nor of experimental artificial origin (6). Anti—phosphatidylserine antibodies have also been used in cancer therapy it is suggested that the ’ (7). Hence, SA-bearing liposomes may prove to be ive in ancer therapy. Since the drug free liposome itself selectively recognizes and destroys elevated PS exposed on membrane surfaces, this property of the liposome can be utilized in anticancer therapeutic gies] The SA-bearing liposome has been shown to cause immunomodulation in the host and targets PS-bearing parasites for ction (8). It is t that the SA—bearing liposomes should effectively target the cancer cells.

Moreover, the efficacy ofthe ncer drugs which target the surface PS ofthe tumor cells and also otherwise shall profoundly increase when stered entrapped in these liposomal formulations. The anticancer drugs frequently. affect the-normal cells and thus cause severe side effects. But, whenadministered in a liposomal covering shall thus decrease the cytotoxicity. We can also ensure ive and selective targeting ofthe anti-cancer drugs when administered within this SA-‘bearing me because PS on the tumor vessels is abundant and is on the luminal surface ofthe tumor endothelium. This renders it directly accessible for binding by any vasculature * targeting agents in the blood. Moreover, it is present on a high percentage of tumor elial cells in diverse solid tumors; and it is absent from endothelium inall normal tissues examined to date, thus ensuring selectivity. The phenomenon of PS exposure is not exclusively associated with apoptosis. For instance monocytes entiating into macrophages and a'subpopulation of T-lymphocytes expose PS.

Living tumour cells and endothelial cells ofthe tumour blood vessels also express high levels of PS on their surface. Furthermore cell surface exposure of PS is independent of cell type and thus independent of the type of cancer (3). Vascular-targeted strategies directed against d PS may be a powerful t to postoperative chemotherapy in preventing relapses after cancer surgery (9). lt has also been shown that B]6Fl0 murine melanoma cells express high ievels of PS on their surfaces (10). These features support the proposal that PS can be utilized as an attractive target for the tumour blood vessels as well as the living tumour cells. mes are under extensive investigation as targeted drug delivery against many diseases because the drug here is protected from ironment and thus remains stable for a much longer time- They are small concentric bilayered vesicles, in which an aqueous volume is entirely enclosed by a membranous lipid bilayer mainly composed of natural or synthetic phospholipids. The first tion of liposomes, a.k.a. conventional liposomes, was developed in the early 70’s. They were composed of phosphatidylcholine, phosphatidylserine, phosphoglycerol, and cardiolipine ated with cholesterol J so as to reduce permeability (11).

Liposomal drug ry : Lipid associated formulations have been proved to be more effective therapies with much lesser toxicity against in vitro and in viva anti-protozoan activity e.g. visceral leishmaniasis (VL). The advantage of such formulations ‘is their ability to concentrate high levels of drugs in the infected target " organs. For example, AmBisome, a liposomal formulation of ericin B, is the safest and can be administered at doses much higher than the free form of the drug with much less toxicity (12). The drug-free stearylamine (SA)-bearing cationic liposomes have in vitro activity. A single dose of the drug entrapped in this cationic liposome formulations has a synergistic activity and hence a much more profound effect on the . The profound effect ofthe drug-free liposome is due to recognition of the phosphatidylserine (PS) on the parasite membrane by the SA mes. The study of the mode of action of the SA-bearing cationic liposomes revealed that the recognition ofthe surface PS by the liposome is necessary for its ability to damage the te membrane resulting in its ultimate death (12). This peculiar mode of action of the drug—free liposomes leads to an interesting'hypothesis. SAzPCzChol in its molar ratio 1:415 was found to be toxic for cancer-derived and normal human cultured cell lines at varying degrees (11). Cationic liposomes are used as a delivery system to cells of compounds capable ofsilencing a target protein and enzyme substrate and also used for detecting inhibitory activity of a target protein in a cell as well as signal uction pathway in a cell (15).

Classification of Liposomes: The properties of liposomes vary substantially with lipid composition, size, surface charge and method of preparation. Liposomes can be either classified following in the following mannerzv ’10 ing to size or lamellarity Small Unilamellar Vesicles (€ 100 nm, a.k.a. SUV) are surrounded by a single lipid bilayer of 25-100 nm diameter. Large Unilamellar Vesicles (100 — 500 nm, a.k.a. LUV) are a heterogeneous group of vesicles similar to d are surrounded by a single lipid bilayer. Finally, Multilamellar Vesicles (> 500 nm, a.k.a. MLV) consist of several lipid bilayers ted from each other by a layer of aqueous solution. They have onion like structures (11).

According to on the method of preparation Reverse Phase Evaporation Vesicle (REV): The reverse—phase evaporation que, the first to use —in—oil’ emulsions, encapsulates up to 50% of . Preparation of e—phase evaporation vesicles (REV) consists of a rapid injection of aqueous solution into an organic solvent which contains the lipids dissolved. Thus, following the formation of water droplets (‘water-in-oil’ emulsion) by bath sonication of the tWo- phase mixture, the emulsion is dried down to a semi-solid gel in a rotary ator.

The next step is to subject the gel to vigorous mechanical shaking to induce a phase change from a in-oil emulsion to a vesicle suspension. In these circumstances, some water droplets collapse, and these droplets attach to adjacent, intact vesicles to form the outer leaflet of the r of a large unilamellar liposome (diameter in the range of0.l to l um) (16).

Dehydration-Rehydration Vesicles (DRV): Another method that produces ation-rehydration vesicles (DRV) is both simple and easy to scale up, and usually; gives high yields of solute entrapment (up to 80%). Preparation of DRV consists of mixing an aqueOus solution of the solute with a suspension of "empty’ (water- containing) liposomes and freeze—drying the resultingmixture. The intimate contact of flattened liposomal membrane structures and solute molecules in‘ a dry environment and the fusion of membranes caused by dehydration facilitates the' incorporation of solute during the controlled rehydration steps. tion of solute-containing DRV from ‘unentrapped solute can be d out by centrifugation easily if needed. Vesicles formed by the dehydration—rehydration technique are multilamellar with heterogeneous sizes (diameters varying from 0.1 to 2.0 um) (16).

Multilamellar Vesicles (MLV): The most easily prepared and processed liposomal‘ form is Multilamellar vesicles or MLVs. MLVs are prepared by first casting a lipid film in organic solvent (chloroform). Then lipid particles are dispersed in aqueous t '15 followed by probe sonication. This form of liposome has advantages over both DRV and REV form of liposome due. to its optimum size (200—250nm) and multilamellar structure which allows higher amount of drug to be successfully entrapped into the me. The MLV form of liposome showed maximum efficacy in the present model and t drug entrapment efficacy. Hence, most ofthe ments were performed with this particular form of liposome.

According to in viva application: Conventional liposomes which may be neutral or charged. They are usedmainly for macrophage targeting or for vaccination. _ negatively Stealth ically stabilized) liposomes which carry polymer coatings to obtain prolonged circulation times. lmmunoliposomes (antibody targeted) are used for specific targeting and ic liposomes are employed mainly for gene delivery or cancer therapy. mes containing‘cationic lipids (DOTAPzDOPE, SA) have been widely used as transfection ors both in vitro and in vivo due to their ability to interact with negatively d molecules such as DNA and phosphatidylserine of cell membranes. The amphiphilic properties of cationic lipid molecules together with positive charge and defined phase behaviour of liposomes composed of them, make possible interactions such as adsorption, fusion, poration and destabilization with negatively charged membranes. Some cationic lipids are able to penetrate natural membranes and localize in, the inner leaflet, forming invaginations and even endosome- like vesicles. The initial event oecurring between cationic liposomes and negatively charged plasma membrane is adsorption. Electrostatic and hydrOphobic ctions may lead to hemifusion, fusion, poration or, alternatively, receptor- mediated endocytosis may occur (17). brane mimetic model system were prepared with PC:PS, PC:PA or PCIChol ‘ liposomes and it was found that PC:SA liposomes had specific affinity for PC:PS liposomes rather than PC2Chol or anionic PC:PA liposomes, This further supported the PC:SA liposome cted mainly with PS. This indicates that the SA-bearing liposomes can be used as a valuable delivery system against cancer cells which also have elevated levels of negatively charged PS on the outer surface of their membranes (12). Even in cationic mes variations can be made on the basis of methods of preparation. Previously, work has. been done on comparative study of these different types of liposomes and their n entrapment efficiency with Leishmania donovam' promastigote membrane antigens (LAg). In that study mice were immunized with LAg encapsulated in multilamellar vesicles (MLV), dehydration—rehydration vesicles (DRV) and reverse-phase ation vesicles (REV) and were challenged with parasites ten days after vaccination. Leishmanial n '(LAg) in MLV or DRV induced almost te‘protection, while LAg alone or entrapped in REV exhibited partial resistance.

MLV encapsulated LAg trated durable cell —mediated immunity and mice challenged ten weeks after vaccination could also resist experimental challenge strongly (18).

Selecting an Appropriate Antineoplastic Agent: Most patients with advanced solid tumours still die of their disease. For this reason, new effective drugs are .

A number cancer drugs are under igation at present which target the tumor cell at DNA or protein level. Other elements interacting with tumors like the endothelium or extracellular matrix may also be targeted.

Camptothecin (CPT) is a quinoline alkaloid isolated from the bark and stem of Camptotheca acuminata (Camptotheca, Happy tree), a tree native to China (19). It ts potent xic activity against a range of tumor cell lines CPT, possesses a high melting point (264-267°C), and has a molecular weight of 348.1] obtained by high-resolution mass spectroscopy, corresponding to the formula (C20H16N204).

'Camptothecin inhibits both DNA and RNA synthesis in mammalian cells. The inhibition of RNA synthesis results in shortened RNA chains and is y reversible upon drug removal while inhibition of DNA synthesis is only partially reversible. CPT binds to Topoisomerase l and DNA complex (with Hydrogen bonds), resulting in a stable ternary complex. Topoisomerases are a family of enzymes which relax supercoiled DNA by making transient single stranded breaks in the DNA, allowing the uncut strand to pass through the break before rescaling the nick (thus increasing its linking number by 1.). Binding ofCPT ts this rejoining step which ultimately leads to DNA fragmentation-and apoptosis. This drug is widely distributed in the body including the central nervous system, lungs and liver (20).

Camptothecin encloses in its structure a highly ated pentacyclic ring with an a-hydroxylactone portion at carbon l2 which is essential for its in vitro and in vivo antitumor activity. Unfortunately this lactone ring is highly susceptible to hydrolysis and under physiological conditions i.e., at pH 7 or above, the lactone ring readily opens to yield the inactive carboxylate form ot‘the drug (20). Ring opening of camptothecin is thought to result in a loSs of activity due to the ing three s First, the carboxylate form displays sed association with the membrane. .25 Second, ring opening results in a charged drug species which exhibits d dit‘l‘usibility through lipid bilayer domains. Third, evidence from cell-free experiments indicates thatring opening results in cantly reduced intrinsic potency towards the topoisomerase—l target.

The above drawback along with poor water lity and high e drug on limits the application of CPT in therapeutics. For this reason different lipid based formulations were investigated and it was found that CPT is soluble in various lipids and also biologically active at the same time. Two types of spectroscopic data are ble which support that liposome associated camptothecin is stable. The first evidence comes from, where there is a blue shift in the drug’s emission spectrum which ‘is observed upon its association with membrane. Such a al shift is indicative of alchange in the dielectric constant of the medium surrounding the fluorophore, as when a compound leaves an aqueous environment and intercal'ates in between the lipid acyl chains (21). Thus, liposomal drug delivery s are of, ' potential utility for introducing camptothecin (or d lipophillic analogues) in its stable and pharmacologicallyactive form to cancer victims.

.Doxorubicin (DOX) is another an important class of drug that is used in cancer y. It is an anthracycline antibictic. It is photosensitive in nature. It was first extracted from Stieptorhyces peucetius. lt is used in the treatment of several cancers that include breast cancer, lung , ovarian,-gastric, Hodgkin’s-and Non Hodgkin’s lymphoma, Multiple myeloma and sarcoma and pediatric cancers. The main function ofdoxorubicin is in int'ercalating DNA. There are two proposed mechanisms by which bicin acts on cancer cells. They are (i) alation‘into DNA and disruption of topoisomerase ll mediated DNA repair and (ii) Generation of free radicals and their disruption cf cell membrane and damage to DNA and proteins. In other words doxorubicin is oxidized to semiquinone (an unstable. metabolite) which is converted back to doxorubicin in a process that releases reaCtive oxygen species (ROS). ROS have the ability to cause lipid peroxidation and membrane damage, DNA damage, Oxidative stress, and stimulates apoptic pathways of cell death. Doxorubicin can enter the nucleus, poison DNA omerase H and cause damage to the DNA and cell death. A major limitation for the use ot‘doxorubicin is cardiotoxicity, with the total cumulative dose being the only criteria currently used to predict the toxicity (22).

] The present invention claims that cationic liposome in all its three forms (MLV, - DRV, REV) is a potential anticancer agent which selectively s the cancer cells through the cancer cell surface exposed phosphatidylserine. The formulation has no e effects on the normal cells and hence is a successful targeted therapy against cancer. The ncer drug ulated formulations had significant anticancer effect both in vitro and in vivo. The drug encapsulated liposome has a synergistic effect and hence shall be effective in bringing down the dosage of the drug and thus protecting against chemotherapeutic toxicity. The liposomal formulation is successful in controlling the e chemotherapeutic effect of anti-cancer drugsdue low dosage of application with high efficacy and ed delivery with minimum damage to normal cells. The formulation mainly works through the sic kinase signalling pathway of the cancer cells. It can be, a valuable therapeutic agent since it showed negligible therapeutic toxicityvand miraculous therapeutic ency in vivov.

OBJECTIVES OF THE INVENTION id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12" id="p-12"

[00012] The ‘main objective of the present disclosure is to provide a synergistic liposomal ation comprising of PC: SA and anticancer drug.

Another object of the present disclosure is to provide a formulation comprising: 'phophatidylcholine, stearylamine and camptothecin in a molar ratio of 7(PC): 2(SA): 0_.7(CPT). id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14" id="p-14"

[00014] Yet another object ofthe present disclosure is to provide a formulation prepared in the form of dehydration-rehydration vesicles (DRV), reverse phase evaporation vesicle (REV) and multilamellar vesicles (MLV).

Still another object of thelpresent disclosure is to provide the use of phosphatidylchpline (PC) and stearylamine (SA) in a molar ratio of 7:2, for the ent of cancer.

Another object of the present disclosure is that PC: SA liposome-induced killing of cancer cells is due to apoptosis, dissipation of mitochondrial membrane potential, increased level of ve oxygen species (ROS).

Yet another object of the t disclosure is to reduce the side effect the drugs thus improving their therapeutic efficacy.

Y OF THE ION [00017a] In one aspect there is provided use of a mal formulation for the manufacture of a medicament for the treatment of cancer, wherein said liposomal formulation comprises phosphatidylcholine (PC) and stearylamine (SA) in a molar ratio of 7:2.

Accordingly, the present disclosure provides, a synergistic liposomal formulation for the treatment of cancer wherein the ation comprises, tidylcholine, stearylamine and anticancer drugs.

In an embodiment of the present disclosure, the anticancer drugs are selected from the group consisting of camptothecin (CPT), doxorubicin (DOX), cisplatin or paclitaxel.

In still another embodiment of the present disclosure, the ratio of phatidylcholine, stearylamine and camptothecin in synergistic liposomal formulation is 7(PC): 2(SA): 0.7(CPT).

In yet another embodiment of the t disclosure, the ratio of phatidylcholine, stearylamine and doxorubicin in the synergistic liposomal formulation is 7(PC): 2(SA): 0.5(DOX).

In still another embodiment of the t disclosure, the formulation is prepared in the form of dehydration-rehydration vesicles (DR V), reverse phase evaporation vesicle (REV) and multilamellar es (MLV).

In yet another embodiment of the present disclosure, the formulation is used for the treatment of murine melanoma, rat glioma, colorectal adenocarcinoma, human colon carcinoma, chronic myelogenous leukemia, acute lymphoblastic leukemia and ascites oma in vitro. (26575770_1):RTK 10 ln still another embodiment of the present disclosure, the dose ofthe synergistic liposomal ation is used at 20—140 ug/ml with respect to PC.

Yet another embodiment of the t disclosure provides the use of liposomal formulation for the treatment of , wherein the said formulation ses of phosphatidylcholine (PC) and stearylamine (SA) in a molar ratio of7z2.

Still another ment of the t disclosure, the ECSO value of the liposomal formulation against cancer cell lines is in the range of 60-80 pg/mlJn yet another embodiment of the present disclosure, the dosage ofthe liposomal formulation is administered at 3 doses of 800 mg /Kg body weight (intravenously) with respect to PC against DEN induced carcinoma in rats.

In still another embodiment of the present disclosure, the dosage of the liposomal formulation is administered at a single. shot injection of L7 g/Kg body weight with respect to PC (intravenously and intra—peritoneally) in Swiss albino mice to inhibit growth of Ehrlichs ascites oma (EAC).ln yet another embodiment of the present disclosure, the dosage of the liposomal ation is administered at a single dose of 7 mg/Kg body weight (subcutaneously) with respect to 'PC against Bl6FlO melanoma in vivo. ln yet another embodiment of the present disclosure, the anti-cancer drug CPT is administered at a single dose of 350 rig/Kg body weight entrapped in 7 mg/Kg body weight ofthe liposomal formulation with respect to PC (subcutaneously) for increased anti-tumor effect ofthe free liposome against B l6Fl>0 melanoma in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS: Figure l: In vitro cytotoxic effect of graded doses (20-l40 ug/ml with, respect to PC)‘of cationic (PC:SA), neutral (PC:Chol) and anionic (POPS) liposomes on different cell lines (SXIOS/ml) after 2 h of treatment. , ‘ rat brain astrocytes, rat C6 glioma, SW480, HCTl l6 and HepGZ cell lines were cultured in DMEM medium supplemented with 10% FCS with graded concentraions of different liposomes. RAW 264.7, K562, MOLT4, U937 cell lines, Ehrlichs ascites oma cells and human PBMC of healthy donors were cultured in RPMI medium supplemented with 10% FCS with graded concentrations ofdifferent liposomes. The cell lines were treated with MLV form of liposomes except where mentioned. BléFIO cells were treated with PC:SA (MLV, REV and DRV), PCzChoi (MLV, REVand DRV) liposomes (A). RAW 264.7 cells were treated with PC:SA (MLV, REV and DRV) liposomes (B). K562 cells were treated with PC:SA, PC1Chol and PCIPS liposomes (C). U937 cells (D), Rat C6 glioma (F), rat brain astrocytes obtained from cerebral cortex of zero day old rats cells (G) were treated with PC:SA and l liposomes. Human PB MC of normal donors (E), Ascites- carcinoma cells obtained from mouse peritoneal fluid (H), SW480 (l), HCT _l 16 (J) and HepG2 cells (K) were treated with PC:SA liposomes. The viability of all the cell types was measured by inhibition of 3-(4, 5—dimethylthiozol—Z-yl)-2, 5- diphenyltetrazclium bromide (MTT) redUction to insoluble formazan by mitochondrial dehydrogenase. Error bars denote standard deviation of 3 ments.

] Figure 2: In vitro cytotoxic effect of graded doses (20—140 ug/ml with respect to PC) of MLV form of PC:SA and PCzChol liposomes On different cells (SXIOS/ml) after 4 h of treatment. U937 cells (B), human PBMC of healthy donors (D) were treated with PC:SA and PC:Chol liposomes. K562 (A), MOLT4 cells (C), rat brain astrocytes (E), rat C6 glioma (F) and Ehrlichs ascites carcinoma (G) pG2 cells (H) were treated with PC:SA liposome. The viability ofall the cell types was measured by inhibition of 3-(4, >5-dimethylthiozolyl)-2_,5-diphenyltetrazolium bromide (MTT) reduction to insoluble formazan by mitochondrial ogenase. Error bars denote standard deviation of3 ments.

] Figure 3: Effect of ty of PC:SA liposome after blockirig it with different '25 doses of PCzPS and PCzChol liposome. PC:SA liposomes (120 ttg/ml with t to PC) were incubated for 30 min with different concentrations of PCzPS or PCrChol liposomes (l5-240 ug/ml with respect to PC) prior to incubation with SXIOS/ml of BléFlO (A), K562 (B), U937 (C) and rat C6 glioma cells (D)‘.After 120 min post— treatment the viability was ed by MTT assay. Data points represent the mean of triplicate samples 3: SEM from a single experiment, representative of three ent experiments.

Figure 4: Blocking of killing activity of PC:SA liposome by annexinVV. Cells were incubated with or without purified recombinant annexin V (5 ug/leOs cells) for 30 min in annexin V binding , washed with 20 mM PBS and resuspended in respective media ented with l0% FCS. K562 cells were treated with 140 ug/ml of PC:S’A liposome with t to PC (A) and Bl6FlO cells were treated with graded concentrations (20—l40 ug/ml) of PC:SA and PCzChol liposomes (B). Cells were washed with 20 lel PBS and resuspended in DMEM medium suplemented with 10% FCS. The viability ofthe cells was determined by MTT assay after 120 min. v [00033] Figure‘S: Comparison of the effect of camptothecin (CPT) ped in PC:SA liposome with respect to PC (20-140 pg/ml) and free CPT (l-7 ug/ml, same concentrations that were entrapped in the liposomes) for.2 h on Bl6FlO, EAC, human PBMC, SW480, U937 and rat C6 glioma cells (5X105/ml). All cell lines were treated with MLV fOrm of PC:SA liposome except where mentioned. Bl6FlO cells were treated with graded doses of CPT entrapped in MLV, DRV and REV forms of PC:SIA liposomes (A). EAC (C), rat C6 glioma (E), human PBMC of healthy donor (G), SW480 (l) and U937 (K) cells were treated with graded doses of CPT entrapped in PC:SA liposome. Bl6FlO (B), EAC (D), rat C6 glioma (F), human PBMC of healthy donor (H), SW480 (.l) and U937 (L) were treated graded doses of CPT. The viability was measured by MTT assay. [[00034] Figure 6: Comparison of the effect of doxorubicin (DOX) entrapped in MLV PC:SA liposome with respect to PC (20-l40 pg/ml) and free DOX (l-7 ug/ml, same concentrations that were entrapped in the liposomes) for 2 h on U937, rat C6 glioma and EAC cells (SXIOS/ml). U937 (A), rat C6 glioma (C) and EAC (E) cells were treated with graded doses of DOX ped in PC:SA liposome. U937 (B), rat C6 glioma (D) and EAC (F) cells were treated with graded doses of DOX. The viability was measured by MTT assay.

Figure 7: Apoptotic effect 0fPC:SA liposome (140 ug/ml with respect to PC) on different cell lines (5X105 cellsfml). A: Bl6Fl0 cells- untreated (3), treated for 2h U1 (b). B: K562 cells— untreated (3), treated for 2h (b) and 4h (c). C: U937 cells— untreated (a), treated for 2h (b). D: MOLT4 cells- untreated (a) and treated for 4h (c). E: Human PBMC from healthy donor- untreated (a) and treated for 4h (b). Annexin V—Pl binding assay Was then performed using annexin V-FlTC apoptosis detection kit followed by analysis in a flow cytometer. Values in the nts represent percent positive’cells. id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36"

[00036] Figure 8: Depolarizatitm of mitochondrial membrane potential. Effect of PC:SA (140 pg/ml. with respect to PC), CPT (7 pg/ml) and 7 ug/ml CPT entrapped in l40 [lg/ml of PC:SA liposomes on different cell lines for 2h treatment. A: B'l6FlO cells- untreated (a), treated with CPT (b), PC:SAF(c) and CPT entrapped in PC:SA ‘liposomes (d). B: K562 cells- ted (3), treated with PC:SA liposome (b). The cells after treatment were stained with the mitochondrial membrane potential-sensitive dye JC—l and analyzed in a flow cytometerrThe tage of cells expressing green and red scence is indicated.

] Figure 9: DNA cell cycle analysis (apoptotic nuclei [Sub GO/GI peak] and Gg/M arrest) by flowcytometry using Pl stain. Different cell lines (SXIOS/ml) were treated with l40_ ug/ml of PC:SA with respect to PC, washed twice in PBS. ed cells Were fixed in 70% cold ethanol and incubated overnight at —200C. After two washes in PBS the cells were resuspended in 0.5 ml of Pl containing RNaseA and the mixture was ted for 20 min in the dark at room ature. The fluorescence ity of Pl was analysed with a FACS Calibur flow cytometry and ‘Cell Quest software. A: Bl6Fl0 cells- untreated (a) and treated for 2h (b). Gates were set to assess the percentage of dead sub GO/G, (P4), Go/‘Gl (P5), S (P6) and (33M (P7). B: K562 cells- untreated (a) and treated for 2h (b). Gates were set to assess the percentage ofdead Sub Go/Gl (P5), (in/G. (P2), S (P4) and GzM (P3). C: U937 cells- untreated (a), treated for 2h (b) and 4h (0). Gates were set to assess the percentage of dead Sub (Jo/GI, GO/Gl, S and Gz/M arrest. D: MOLT4 cells-untreated (a), treated for 4h (b). Sub GO/G] (P4), (So/G] (P5), S (P6) and 62M arrest (P7). E. RAW 264.7 cells—untreated (a), treated for 2h (b). Sub Go/G. (P4), GO/G; (P5), S (P6) and (33M (P7). F: Human PBMC of healthy donor-untreatedl(a), treated for 2h (b). Sub GOXG. (P4), GO/G] (P5), S (P6) and (32M (P7). Bars denote boundaries ofcell cycle.

Figure ‘10: Determination of PC:SA liposome induced ROS generation. K562, MOLT4, U937 and normal human PBMC were treated with 40 and [40 ug/ml of PC:SA with respect to PC for 4h and Bl6FlO and RAW 264.7 cells were treated with same concentrations of PC:SA me for 2 h (A), K562 cells were treated with graded concentrations of PC:SA liposome (20-l40 ug/ml) for 2 h (B). ROS was measured in treated and untreated cells incubated'in the fluorescence dye A (luM) for 30 min at 31°C by fluorescence ophotometer. Data are expressed as mean of: SEM ofthree independent ments. id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39" id="p-39"

[00039] Figure 11: PC:SA' liposome induces caspase dependent cell death. K562 cells were treated with graded doses ot‘PC:SA liposome (20—140 ug/ml with respect to PC) in the presence or absence of pancaspace inhibitor fmk (lOuM). The reduction in the viability of cells was determined by MTT-assay. Data points ent the mean oftriplicate s :‘SEM from a single experiment. id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40" id="p-40"

[00040] Figure 12: Transmission electron microscopy of U937 cells treated with PC:SA liposomes. U937 cells were inci‘ibated for 4 h under standard ions with medium alone (a), 40 ug/ml (b) and I40 rig/ml (c) of PC:SA liposomes with respect to PC.

Three representatives of on micrographs of untreated (a), 40 ug/ml of PC:SA treated (b) cells revealed disruption of membrane integrity, and I40 ug/ml of PC:SA treated (c) extensive vacuolization and membrane breakage (as well as depletion of electron—dense cytoplasmic material indicating that cell death is in process. Seale bars: 2000 nm (a and b), 1000 nm (c).

Figure l3: lmmunoblot—based demonstration of the ement of p21 (a), ERK (b), p-ERK (c), caspase 8 (d), cleaved caspase 9 (e) and cleaved caspase 3 (t) in PC:SA (l40 pg/ml with respect to PC) treated RAW264.7 (left column) and Bl6FlO (right column) cell lines. B-actin was used as g control (g) (A). lnvolvement of p- ERK (top panel) and Bid (middle panel) in PC:SA (20-140 pg/ml with respect to PC) treated U937 cell line B-actin was used as loading control (bottom panel) (B). 2h treated were harvested, lysed, and equivalent amount of lysates were separated by SDS-PAGE and electrotransferred. The membranes were probed With anti—ERK, anti~ phospho-ERK, aspase 8, anti—cleaved caspase 9, anti-p2], anti-Bid and anti-B Actin antibodies.

Figure 14: lmmunoblot~based determination of p-Pl3K expression. K562 cells were treated with I40 pg/ml of PC:SA liposome with tto PC for 90 and l20 min, harvested, lysed and equivalent amount of lysates were separated by SDS-PAGE and electrotransferred. The membranes were probed with hospho-PI3K antibody (top panel). B—Actin was used as loading l (bottom panel).

Figure l5: Effect of administration of 60 mg of PC:SA liposome with respect to PC on growth of EAC cells (2X106) (i.p) in Swiss albino mice. Untreated EAC injected mice (A). Mice treated with PC:SA me (i.v) on day 2 after EAC injection (B). Mice treated with PC:SA liposome (i.p) on day 2 after EAC injection (C). Animals were sacrificed on day I4.

Figure 16: To assay the effect of PC:SA and CPT-entrapped PC:SA liposome on the tumor development in syngenic C57BL6 mice ctive and therapeutic aspect). 2Xl0(3 Bl6FlO cells preteated for 2 h with 140 pg of PC:SA and 7 pg CPT entrapped in MO pg PC:SA liposomes (with respect to PC) were injected in C57BL6 mice subcutaneously. Animals ed with untreated Bl6FlO cells were kept as control (protective aspect) (A). Bl6FlO cells were injected on day 0 and treated on day 2 with either 140 pg of PC:SA liposome alone or with 7 pg of CPT entrapped liposome. Mice injected with Bl6FlO cells and left untreated served as control (therapeutic aspect) (8). Animals were sacrificed after 21 days and tumor s were observed.

] Figure l7: Histological and hemical examination of liver sections of experimental rats. Eosin—haematoxylin stained liver ns of control rats showing (J1 normal liver architecture, DEN treated control showing (A dialated hepatic veins, DEN treated control, showing hyperplastic nodulesH and DEN+ PC:SA liposome treated, g architecture similar to normal liver with some amount of periportal fibrosis (-' ). Scale Bar: 200 pm.

Figure 18:. Effect of, PC:SA liposome treatment on serum biochemical ‘ parameters in DEN—induced hepatotoxicity in rats. At the end of 18 week starting from the lst day of DEN administration, blood was collected from heart in the rats of-each group. Serum ate transaminase (AST), alkaline phosphatase (AP) and serum e transaminases (ALT) were determined using a standard kit manufactured by Span Diagnostics Ltd. Values are sed as mean i SEM. id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47" id="p-47"

[00047] Figure l9 Comparative study of free PC:SA liposome and free anti-cancer drugs: The EC50 values of free PC-Sa liposome (a) and free anti—cancer drugs DOX or irinotecan hydrochloride against cancer cell lines treated for 2 h.

Table l: Size and Polydispersity index ofliposomes.

Table 2: Percentage of CPT entrapped in PC:SA REV, DRV and MLV liposomes.

Table 3: Percentage of DOX ped in PC:SA MLV liposome.

Table 4: Effect of single shot injection of 60 mg of PC:SA liposome 0n EAC cell number and volume of fluid in the peritoneal cavity of Swiss albino mice injected with EAC cells. Values are expressed as mean :t SEM. id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52" id="p-52"

[00052] Table 5: Growth inhibition of Bl6Fl0 melanoma by the subcutaneous injections of Bl6Fl0 cells pre—treated with I40 pg of PC:SA and 7 pg of CPT entrapped in 140 pg of PC:SA liposomes with respect to PC (protective aspect).

Table 6: Growth inhibition of B|6Fl0 melanoma by the aneous injections of MO ug/mlof PC:SA and 7pg of CPT entrapped in _l40 ug of PC:SA liposomes with respect to PC in C57BL6 mice previously injected with Bl6FlO cells (therapeutic aspect). Values are expressed as mean i SEM. id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54" id="p-54"

[00054] Table 7: Effect of PC:SA liposome on % ofincrease in RLW in DEN induced hepatocellular carcinoma. At the end of IS week starting from the lst day of DEN administration final liver weights of all animals were recorded and RLW were calculated. Values are expressed as mean 3: SEM.

DETAILED DESCRIPTION OF THE INVENTION The following iations will be employed: PO phosphatidylcholine SA- stearylamine PS— atidylserine id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59" id="p-59"

[00059] Chol— cholesterol MLV- multilarnellar vesicles DRV— dehydration—rehydration vesicles ] REV- e phase evaporation vesicle CPT- camptothecin id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64" id="p-64"

[00064] DO‘X- doxorubicin PBS- Phosphate buffer saline DDAB- ldimethylammonium bromide DOTAP- [l-(2,3-Dioleoyloxy)]-N,N,N—trimethylamonium propane methylsu l fate PBMC— peripheral blood mononuclear cell RPMl- Roswell Park Memorial Institute medium FBS— fetal bovine serum [0007.1] MTT- 3-(4, S—dimethylthiozol—Z-yi)—2, enyltetrazolium e FACS- fluorescence—activated cell sorting Pl-propidium iodide FlTC- fluorescein ocyanate Aw- mitochondrial ne potential JC-l- 5,5',6,6’-tetrachlor0~l,l',3,Y—tetraethylbcnzimidazolocarbocyanine Iodide id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77" id="p-77"

[00077] ROS- reactive oxygen species H2DCFDA— 2’,7’-dichlorodihydrofluorescein DCF- dichlorofluorescein TEM— Transmission Electron Microscopy' Pl3—Kinase— phosphatidylinositol-3—kinase' .15 ] MAP Kinase- 'mitogen-activated protein kinase ERK- extracellular signal-regulated kinases EAC-Ehrlichs ascites carcinoma HCC— hepatocellular carcinoma DEN- diethvlnitrosamine id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87" id="p-87"

[00087] RLW- relative liver weights H&E- hematoxylin and eosin AP- alkaline atase AST— serum aspartate transaminase A-LT- serum alanine transaminases The present disclosure. provides liposomal ation comprising of PC:SA in three forms MLV, DRV and REV in 7:2 molar ratio. In our study we compared the cytotoxic activity of 'stearylamine-bearing ic liposome against normal cells (human PBMC, murine macrophage andirat brain astrocytes) and nine different cancer cell lines (three human leukemia, one murine melanoma, two human colorectal a'denocarinoma, one rat glioma, and one murine ascites carcinoma cells). We observed that there was a dose-dependent ive killing of these cancer cell lines by PC:SA mes and that this killing activity correlated with the exposure of vely- charged phosphatidylserine (PS) on the surface of these cell. PC:SA me was non—toxic for normal cells like human PBMC and murine macrophage cell line. Neutral (PCzChol) and negatively charged (PCzPS) liposome was found to be non-toxic for both cancer and normal cells. Another cell line called human liver- hepatocellular carcinoma showed very less killing effect by PC:SA lipoSome which could be due to very less PS exposure on this cell line. To reconfirm the PS—specific affinity of SA- bearing liposome; PC:SA liposome was preincubated with liposome composed of PCzPS. Killing activity of PC:SA liposome-was inhibited with gradual increase in the dose of PCgPS' liposome. Killing activity of liposomeswas also inhibited at 2 hr treatment after ocking the cells with annexinV. igation to understand the ism of induced killing of cancer cell lines revealed that cell death was due to sis. This‘was determined by the increase in appearance of PS on the external surface of the plasma membrane (binding to annexin V). Dissipation of mitochondrial transmembrane potential (A‘l’m), a characteristic feature of apoptosis, was ' observed in cells treated with PC:SA. Our data show thatl’CSA me ' enhanced the levels of reactive oxygen species (ROS). To investigate whether caspase has any role in cell death, cells were preincubated with Z-VAD-fmk, a broad spectrum caspase inhibitor, before PC:SA treatment. There was reduction in the killing activity revealing the caspase dependent mode of cell death. Cell cycle analysis reveals that ent of cancer cells with PC:SA causes appreciable apoptosis of the cells with accumulation of apoptotic nuclei at the sub GO phase and an arrest of cells at the G2-M phase. involvement of MAPK mediated apoptotic pathway, phosphatidylinositol kinase (Pl3K)/serine/threonine kinase (Akt) signaling pathway and activation of pro- apoptotic protein Bid' were observed in the killing activity of PC:SA liposome against the cancer cell lines. A single sh‘t injection of PCSA liposome inhibits the growth of Ehrlichs asoites carcinoma in Swiss albino mice. The treatment with PC:SA liposome prevented the liver from developing diethylnitro’samine (DEN)-induced in adult male Swiss Albino I hepatocarcinoma rats. Treatment with the ation of Bl6F10 in ic black mice C57BL6J causes preventive and therapeutic effect in animal model. Anti-cancer drugs like CPT and DOX entrapped in PC:SA liposome increases the anti-cancer effect of the free drug and. free liposome against the cancer cell lines. Similarly treatment with CPT entrapped PC:SA ofB l 6F10 in syngenic black mice J increases the anti—tumor effect of the free liposome\in animal model.

Others have previously ed that some cationic antibacterial peptides exhibit a broad spectrum of cytotoxic activity against cancer cells, which could e a new class of anticancer drugs. The anticancer activity of a cationic amphiphilic peptide ABP-CM4, an antibacterial peptide from Bombyx mori, against leukemic cell lines THP—l, K562 and U937 was ted, and the cytotoxicity compared with the effects. on non—cancerous ian cells, including peripheral blood mononuclear cells (PBMCS), HEK—293 and erythrocytes (42). Various tumor cells have elevated surface levels of negatively d phospholipids, i.e. PS. Cytotoxic activity of the in derived peptide NK—2 was ed againSt normal human lymphocytes and seven different cancer cell lines (two neuroblastoma cell lines ‘LA-N—l and SH-SYSY, colorectal adenocarcinoma cancer cells SW480, lymphoma cell line U937, leukemia cell line K562 derived from c myelogenous leukemia, .lurkat and MOLT-4 both derived from acute blastic leukemia). NK-2 which consists of 27 amino acid es with an overall positive net charge and adopts an amphipathic, alpha-helical secondary structure upon membrane interaction. it was demonstrated that NK-Z selectively kills some cancervcells and that this killing activity correlates with the membrane exposure of negatively charge PS on the surface of these cells and the capacity of the peptide to alate into PS-containing model membranes (10).

Patients with cancer are at increased risk for thrombotic complications. Bl6FlO and WM4 ma cells expose PS on their surfaces. The presence of PS is . essential for the assembly of coagulation complexes leading to robust thrombin formation (17). Rat C6 glioma cells support all procoagulant reactions g to robust thrombin formation. This ability results from concomitant tissue factor (TF) * exposure and from the presence of anionic lipid PS at the outer leaflet of cell membrane (18). In the present study, we report that SA-bearing cationic liposomes in .10 7:2 molar ratio have dose—dependent anti-tumor efficacy as proved by its effect on Bl6FlO murine ma cells, K562, U937, MOLT4, rat C6 glioma, SW480, HCTll6- and Ehrlichs ascites carcinoma cells by interacting with the negatively charged PS on the e of the cancer cells. Two types of mes l (PCzChol) and cationic (PC:SA) Were prepared by three ent methods (MLV, DRV and REV). We took two cell- lines: Bl6FlO murine melanoma which expresses considerable amount of PS on its surface and non-cancerous cell line RAW 264.7 murine macrophage. The latter, thus serves as our control cells. In case of K562, MOLT4 and U937 normal PBMC serves as the control cells and in case of rat C6 glioma cell line rat brain astrocytes serve as control. PC:SA liposome also showed anti—tumor activity against two humancolorectalcancer cell lines SW480 and HCTl 16.

The neutral PC:Chol mes did not show significant cytotoxicity on cancer cells whereas the cationic PC:SA ones proved to be very effective in killing these cells Next, we checked if the cationic liposomes could prove to be equally effective in killing RAW 264.7, normal human PBMC and cancerous cell line HepG2. However, they exhibited much lesser cytotoxicity on these cells which can be attributed to their low surface PS t. Thus it Was found that empty ic liposomes had intrinsic killing ability on the cancer cells. It has been seen in previous studies with Leishmcmia parasites that PC:SA liposomes involves specific interaction with negatively charged PS of parasite membrane. resulting in severe damage of the membrane and ultimate death of the parasite (22). Similar mechanism of action of these liposomes might be responsible for killing ofthe cancer cells in our study which is a part of our future plan of work. We wanted to determine the role of SA in PC:SA in the killing se against cancer cell lines. We d the cationic PC:SA me with anionic PCzPS liposome. The PS of POPS liposome blocks the SA of PC:SA liposome. After preblocking, the liposome was used to treat todifferent tumor cell lines (K562, U937 and rat C6 glioma). MTT results te that pre-blocking PC:SA liposome (120 ug/ml) in increasing doses of PCzPS (7:2) liposome effectively decreases the killing efficacy of the liposome, indicating the .role of SA in the killing effect of PC:SA liposome. lt is hypothesized that SA of PC:SA me binds with the PS on the e of the tumor cells and causes the killing effect. To confirm the-PS-specific affinity of SA-bearing liposome, we showed that killing activity of liposomes was also inhibited at 2 hr treatment after pre-blocking the cancer cells (K562 and BloFl0) with annexin V. id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93" id="p-93"

[00093] Apoptosis is by far the best characterized type of cell death and classified as programmed cell death I. It is defined by morphological features ing up of the cell, reduction of cellular 'and nuclear volume, nuclear ntation, plasma ne blebbing and phosphatidylserine exposure, loss of mitochondrial membrane ial, generation of reactive oxygen s (ROS) and caspase activation (43). On treatment with 140 ug/ml of PC:SA liposome, cancer cell lines like K562, U937 and MOLT4 showed a significant increase in the population of the early apoptotic cells indicated by annexin V positive cells. A small number of cells showed annexin V and Pl positive indicating late apoptotic cells too. Normal human PBMC cells ted a small increase in the percentage of early apoptotic cells on treatment with 140 ug/ml of PC:SA MLV. Hence. PC:SA shows selectivity for normal and cancer cells ensuring safety and effectivity. Analysis of mitochondrial membrane potential (Aw), by JG] staining showed that the untreated control cells had a polarized Aw (non-apoptotic, healthy cells). Dissipation of mitochondrial transmembrane potential (ALPm), a characteristic feature of apoptosis, was observed in K562 cells treated with l40 ug/ml of PC:SA. There was a loss in Aw on treatment of Bl6FlO cell line with 7 ug/ml of CPT indicative of apoptosis. However, there was a sharp increase in the Aw rization on addition. of I40 ug/ml of PC:SA MLV on Bl6FlO cell line. This significant increase strengthens our findings that empty cationic PC:SA MLV liposomes are effective apoptotic agents against cancer cells. The depolarization further increased with the treatment of CPT entrapped PC:SA MLV liposomes. This showed that the drug entrapped liposomes had a synergistic effect in lowering the Aw compared to CPT or liposomes alone. PC:SA liposome also increases the level of reactive oxygen species (ROS) in cancer cells. Cell cycle is reveals that * treatment of Bl6FlO and K562 cells with PC:SA causes iable apoptosis of the cells with accumulation of cells at the sub GO phase. Whereas, in case of U937 there is an arrest of cells at the G2—M phase as well as accumulation of apoptotic nuclei at the sub (30 phase and in MOLT4, there is an appreciable arrest of cells at the GZ-M phase.

There is no effect as such on PBMC and RAW 264.7 cells. id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94"

[00094] To determine the molecular ism of the action of PC:SA liposome the K562 cells were pretreated with pan-caspase inhibitor Z-VAD-fmk prior to liposome treatment the killing efficacy of the liposome effectively sed. This indicates that the anti tumor efficacy or apoptosis inducing PC:SA liposome may be d via a caspase mediated pathway. id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95" id="p-95"

[00095] Activation Iof- extracellular —regulated kinases (ERK) stimulates downstream signaling cascades and also modifies transcription causing apoptotic ' changes. Caspase 9 when cleaved gets activated and form part of the apoptosome complex and activate caspase 3 downstream which is also cleaved by PC:SA treatment.

In our experiments we have observed the effect of Pl3K signaling kinase. ement and tion of ERK indicates MAPK mediated apoptotic pathway and egulation , of p-Pl3K indicates phosphatidylinositolkinase (Pl3K)/serine/threonine kinase (Akt) signaling pathway. Involvement of p2l and and» other cell cycle proteins tes the hindrance of cell cycle pathway as a probable ism for PC:SA mediated anticancer effect in some cell types. Our results demonstrate that pro-apoptotic protein Bid is essential for induction of apoptosis of cancer cells by PC:SA liposome." ’When CPT is entrapped in PC:SA liposome in the molar ratio 7(PC): 2(SA): 0.7(CPT) ' it increased the ancer effect of the free iiposome and free CPT against Bl6FlO, EAC, C6 glioma, SW480 and U937 cell lines. In st PC:SA-CPT showed very less effect on human PBMC of healthy donor. The treatment with the formulation (350 ug/kg body weight of CPT entrapped in 7 mg/kg body weight of PC:SA liposome with respect to PC) of Bl6FlO in syngenic black mice C57BL6J increases the anti—tumor 1o. effect ofthe free liposome in animal model.

When DOX is ped in PC:SA liposome in the molar ratio 7(PC): 2(SA): 0i7(DOX) it increased the anti-cancer effect of the free liposome and free DOX against C6 glioma, EAC and U937 cell lines In 1932, Loewenthal and Jahn ((1932) ed the liquid form in the peritoneum of the mouse and named it as ‘iEhrlich ascites carcinoma" due to the ascites liquid. er with the carcinoma cells Ehrlich ascites carcinoma (EAC) has a resemblance with human tumors which are the most sensitive to chemotherapy due to the fact that it is undifferentiated and that it has a rapid growth rate (44). The mouse Ehrlich ascites tumor (EAT) cell also has a negatively charged surface with a measured isoelectric point of about pH 4.0. It seemed possible that sialic acid might be responsible for the negative charge ofthe EAT cell (45). In the present study the potential anti-cancer s of single shot ion (i.v_and i.p) of L7 g/kg‘body weight i.e 60 mg of free PC:SA liposome with respect to PC was tested. The in vivo inhibition of tumour cells growth by PC:SA me might be due to its preferential binding with the negative charges on the tumour cell surface.

Hepatocellular carcinoma (HCC) is one ofthemost common malignant tumors worldwide and the sis still remains dismal (46). Angiogenesis plays a significant role in the aggressiveness of HCC. The only potential curative modality for HCC is y, including transplantation, yet the recurrence rate for this particular cancer is high and len'g—term al rate is rather poor. Both conventional chemotherapy and radiotherapy have been found to be ineffective or only minimally effective in patients with unresectable HCC (41). 3 doses (i.v) of 800 mg/kg body weight i.e 80 mg of PC:SA liposome has proved to be effective inprotecting the rat liver from diethylnitrosamine (DEN) induced altered hepatic functioning, prevented DEN induced hyperplastic nodule formation. Our studies have shown distorted histopathological changes in the liver with formation of hyperplastic nodules'and atypical nuclei on'DEN administration which are indications of DEN induced_ I‘hepatocarcinogenesis. able pathological improvement was noticed in rats- treated with PC:SA liposome.

Through the present work we propose that PS on cancer cells can be utilized effectively as a target for liposomes containing cationic lipids. This unique mode of ion render the SA bearing liposomes valuable drug delivery systems as they are biocompatible and can be administered via various routes. The ncer drugs entrapped in these liposomes are more stable as they are protected from early degradation due to interaction with the biological environment. The side effects of the drugs are also reduced thus improving their therapeutic efficacy, EXAMPLES The following examples are given by way ofillustration of the present disclosure and therefore should not be ued to limit the scope of the presentdisclosure. _ Example] Preparation ofPC:Chol and PC:SA liposomes Preparation of liposomes Multilamellar vesicles (MLV) Liposomes were prepared with PC (20 mg) in association with SA (2 mg) or ' Chol (3mg) (Fluka, rland) at a molar ratio of 7:2. In brief, mes were prepared by adding lipids in chloroform, followed by ating the c solvents to form a thin film in a round-bottom flask. The film was dried overnight in a vacuum dessicator. The film was rehydrated in l ml of 20 mM PBS (pH 7.4), and the suspension was sonicated for 60 s in an onicator, ed by incubation for 2 h at 4°C before using. The concentration of stock solution ofthe mes formed was mg/ml with respect to PC. (22).

Reverse-phase evaporation vesicles (REV): Liposomes were prepared with PC (20 mg) in association with SA (2mg) or with Chol (3mg) (Fluka, Switzerland) at a molar ratio of 7:2, and were dissolved in etherrchloroform:methanol (9:4.2:l v/v/v) in a round ed flask. The film was kept overnight for desiccation in vacuum dessicator. The film was rehydrated with lml PBS and is purged with nitrogen. The mixture was sonicated' to bath sonication at 4°C for 5 min. Organic ts were removed by rotary evaporation at 30 °C for 30 min. .

The flask was again-purged with nitrogen and then kept for incubation in water bath at‘ 45 °C for 30 min. The liposome was then stored at 4 °C. The concentration of stock solution-ofthe liposomes formed was 20 mg/ml with respect .to PC (23).

Dehydration Rehydration Vesicles (DRV): The lipid mixtures of identical molar ratio detailed above were dissolved in chloroform inka round bottomed flask followed by evaporation of the solvent to form a thin film. The film was kept overnight in a vacuum desiccator for desiccation.

‘Rehydration of the film was carried out with lml of sterile distilled water after which it was vortexed and the resulting suspension was sonicated in a bath tion at 4°C for min (at I min intervals). It was then d to stand for 2- 3 h. The mixture was then freeze dried and the powdered e was rehydrated with 0.02M PBS (pH 7.4) in a stepwise manner. Volume of‘PBS equivalent to one-tenth 'ofthe original volume of liposome was added followed by a 30 min interval. This step was repeated twice.

.Finally, the mixture was resuspended properly and PBS was added to make up the al volume. The liposome was stored at 4 °C for 2 h before using (23).

Measurement of vesicle size of liposomes The diameter of the liposomes and the polydispersity index weredetermined by Photon Correlation Spectroscopy (PCS) on Nano Zs Zetasizer (Malvern instruments, Worcestershire, UK) by diluting 1 pl of liposome to 50 pl doubly filtered (0.22 pm pore size) distilled water. Polydispersity index of 0.0 represents a nous particle population s |.0 indicates a heterogeneous size distribution in the me preparations. » Preparation of PC:SA liposomes entrapped with CPT CPT encapsulated REV, DRV and MLV liposomes were prepared by adding DMSO and methanolic solution of] mg/ml CPT to the lipids in the respective organic solvent followed by formation Vofa thin film. The liposomes were processed further as mentioned earlier. The unencapsulated drug was separated from the liposome by two successive washings in PBS by ultracentrifugation (100,000 g, 30 min, 4°C). The - percentage of CPT incorporated in the liposomes was measured spectrophotometrically at 375 nm after dissolving an aliquot ofliposomal preparation in methanol. Finally, the liposomes were stored at 4°C (29).

Preparation of PC:SA mes ped with DOX DOX ' was entrapped in MLV PCZSA liposomes. Briefly, the lipid film prepared with PC- and ic lipid SA at a molar ratio of 7:2 was hydrated with 20 mM phosphate—buffered saline (PBS) containing 1 mg/ml ofDOX and sonicated for 60 ‘ s. Unentrapped drug was washed with PBS through centrifugation at -l ,00,000 X g for 45- min. PC:S/—\ with entrapped DOX was finally resuspended in PBS. For estimation of DOX entrapment efficacy, the liposome was dissolved in l0% Triton-x and measured by Uv-vis spectrophotometry at wavelength of 480 nm. The loading or ' entrapment efficiency is given by the a: ment efficiency (%) = (Encapsulated drug in liposomes/Amount of total drug) XlOO% (48). The concentration of stock solution ot‘all the liposomes formed was 20 mg/ml with respect to PC.

The size and Polydispersity index of PC:SA REV, DRV and MLV mes were ed by Dynamic Light Scattering as shown in Tablel. Thus, in our studies REV was smaller in size compared to DRV and MLV. The spersity index indicates the presence of a heterogeneous population in the Iiposomes prepared by all the three U1 methods.

In our experiment MLV showed maximum CPT entrapment of 100% followed by DRV (88 %) and REV (56 %) (Table 2). The result shows that the percentage of entrapment was t in MLV form of PC:SA liposome and hence we proceeded to do most of the experiments in the particular form of PC:SA liposome. Entrapment efficacy of DOX ' in MLV PC:SA liposome was 100 % (Table 3).

Liposome ‘ Diameter(nm) Polydispersity Index ‘ 3 l05.7i2.89 PC:SA REV PC:SA MLV 202 3 :t 2 94 0.884 . t Table l Liposome Percentage of CPT. entrapped PC:SA REV ’ 56 % PC:SA DRV 88 % PC:SA MLV 100% Table 2 Liposome Percentage of DOX entra ed PC:SA MLV ' Table 3 Example 2 Comparison of in vitro cytotoxic effect of PCzChol and PC:SA (MLV, DRV and REV) and PCzPS liposomes on different cell lines 4] Murine melanoma cell line Bl6Fl0, colorectal adenocarcinoma SW480 cell U1 ‘line, human colon carcinoma HCTl 16 cell line, rat brain astrocytes and rat C6 glioma cell lines were ined in Dulbecco Modified 'Eagle Medium (DMEM) supplemented with [0% fetal bovine serum, sodium pyruvate, 2 mM L-glutamine,' penicillin, and omycin. Murine macrophage cell line RAW 264.7, MOLT-4 cell line (derived from human acute lymphoblastic leukemia), human leukemia cell lines K-562 and human lymphoma cell line U—937 were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, sodium pyruvate, 2 mM amine, penicillin, and streptomycin. The cells were incubated at 37 0C in a humidified atmosphere of 5% C02 in air (10). The Ehrlichs ascites carcinoma cells (EAC) were maintained in vivo in Swiss albino mice, by intraperitoneal (i.p.) transplantation of 15_ cells/mouse every 15 days. Cells were obtained from the peritoneal fluid after days, washed in 0.2M PBS and resuspended in R'PMl medium supplemented with % FBS. al glia-enriched cultures were obtained from cerebral cortex of zero day old rats. The tissues were cleaned free of meningeal tissue, minced, and mechanically dissociated by e through a flame-polished Pasteur pipette.

Dissociated cells were seeded in DMEM with l0% PBS, 4500 mg/liter glucose, l00 U/ml penicillin and 0.1 mg/ml streptomycin. For glia-enriched cultures, the cells were seeded at leO7 in a 75-cm2 flask. The cultures were kept in a humidified chamber at 37°C in a 5% C02 atmosphere for 7 to l4 days, the medium (DMEM with 10% F88, 4500 er glucose, IOO U/ml penicillin, and 0.l mg/ml streptomycin) was changed every 4 days. On the last day, flasks were placed on a shaker platform and shaken at - 220 rpm for 6 h at 37°C to remove the oligodendrocytes and microglia in the cultures.

The glia-enriched cultures were then grown to confluence before use (31).

For isolation of human peripheral blood mononuclear cells from healthy donors (PBMC) heparinized human peripheral blood was obtained from healthy donors and the mononuclear cells (PBMC) were isolated by the density sedimentation on aque-10771‘(400Xg, 30 min at RT) and washed and ended in RPMI 1640 ' supplemented with l0% FCS, 2 ‘lel Lglutamine, penicillin (lOO U/ml), and streptomycin (100 mg/ml) (30). For cell proliferation assay freshly harvested cells (at a density of SXIO5 cells/ml) were seeded in a 96 well cell culture plate, and cells untreated or treated with different concentrations of PC:SA, PCzChol and PCtPS mes 0 ug/ml with t to PC) and tive time points. Inhibition of ' '3~(4, 5-dimethylthiozol—2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction to insoluble formazan by mitochondrial idehydrogenase was used as a viability parameter for the cells and compared with untreated control. After treatment with drug for 2 or 4 h the cells were washed with 20 mM PBS and incubated with 2 mg/ml MTT solution for 4 h at 370C. The reduced formazan was solubilised in DMSO and plates were analyzed by determining the A550 on Thermo MULTISKAN EX plate reader (22).

The potency ‘of PC2Chol (neutral) and PC:SA (cationic) liposomes against murine melanoma Bl6Fl0 cells were determined by MTT assay. Both the Formulations prepared by three different methods i.e. REV, DRV, MLV were used.

PC:SA which is a cationic .lipo'some showed considerable amount of killing of Bl6FlO cells. lt showed almost similar killing effect in all its three forms. REV, DRV and MLV. The percentage of‘viable cells at the highest dose of I40 pg/ml after 2 h treatment were 9.l62 :l: L69 for REV, l4.l9 i 1.95 for DRV and 8.075 i l.76 for MLV. Conversely, the neutral PCzChol counterparts did not show any significant killing even at the t dose of liposome (fig. IA). Therefore, PC:SA liposomes exhibited high cytotoxicity as compared with PCzChol liposomes which are neutral.

We examined the effect of these ic liposomes on murine RAW 264.7 macrophage cells which is a non-cancerous cell line. As evident from the graph, the cationic PC:SA liposomes elicited a much lower killing effect as compared to Bl6Fl0 cell line (fig. lB).

PC:SA MLV cationic liposomes were tested for their ability to kill human ia cells lines K562 and U937 by inhibition of MTT reduction. At a dose of l40ug/ml, the tage of viable cells was only 4% and 20% for K562 and U937 cells (figs, 1C and 1D) respectively after 2 h of treatment. Figs. 2A and B showed that viability further decreased to 0% and i5% for K562 and U937 cells respectively after 4 h of treatment. When MOLT4 cells were d with 140 ug/ml of PC:SA liposome with respect to PC for 4 h the percentage of viable cells was 25% (fig. 2C). In contrast ' control (human PBMC of healthy donor) (figs. IE and 2D) Which was also treated with a similar dose of PC:SA (MLV) liposome and for same durations showed very negligible killing effect. Treatment with PCrChol (MLV)] and PCrPS (MLV) liposomes did not show any g effect on these cell lines even after treatment with the t dose of 140 ug/ml (figs. 1C, ID, 28 and 2D).

Figs. 1F and 2F showed that rat C6 glioma cells were susceptible to the MLV form of the cationic liposome with 32% and 6% cells viable at the highest dose of 140 ug/ml after 2 h and 4 h of treatment respectively-Figs. 10 and 2E showed that PC:SA liposome. was non toxic for non-cancerous cells, rat brain ytes with 99% and 87% cells viable at the higheSt dose of 140 pg/ml with respect to PC after 2 h and 4 h oftreatment tively. Treatment with PCzChol (MLV) liposomes did not show any killing effect on either of these cells even after treatment with the highest dose of 140 ug/ml (figs. IF and 10).

The inhibitory effect of PC:SA (MLV) liposome was also determined by exposure of Ehrlichs ascites carcinoma cells of mouse neal fluid to increasing concentrations ofthe liposome. The highest concentration ofthe liposome (I40 pg/ml) which reduced cell survival by 67% and 77% in 2h and 4 h respectively was determined from the cell survivality curve (figs. lH and 20).

Colorectal adenocarcinoma cells SW480 and human colon carcinoma HCTl l6 Were treated with PC:SA liposome at various concentrations. As seen from figs. ll and ll exposure to increasing concentrations of PC:SA liposome resulted in a dose—dependent inhibition ofthe cells. r cancer cell line 'human liver hepatocellular carcinoma HepGZ had very less killing effect g 87% (fig. lK) and 84% (fig. 2H) viability when ' treated with graded concentrations of PC:SA me for 2h and 4h tively.

Example 3 Identification of the role of stearylamine in anti-tumor efficacy of PC:SA liposomes by observing the effect of PC:SA (MLV) liposome on different cells after pre—blocking the liposome with PC:PS and PCzChol liposomes id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112" id="p-112"

[000112] PC:SA'liposomes (120 ug/ml with respect to PC) were incubated for 30 min with different concentrations of PCzPS liposomes (15-240 ug/ml). The cells were treated with the. pre—incurbated mes for 2 hours. Viability of the cells was d by MTT reduction (22). Figs.’ 3A, 3B; 3C and 3D showed that killing activities of PC:SA liposomes on Bl6FlO, K562, U937 and rat C6 glioma cell lines were inhibited ‘15 with a gradual increase in the dose of. PCzPS liposomes (7:2). Maximum significant inhibition was observed at a concentration of240 ug/ml of POPS. Specificity towards PS was further supported by the negligible effect of the neutral PC:Chol (7:2) liposomes to- inhibit the killing potency ofthe PC:SA liposome. Figure 3A showed that killing aCtivities of PC:SA liposome on Bl6Fl0 cells were unaffected with a gradual increase in PC:Chol liposome (7:2). The result showed appreciable reversal of the effect of PC:SA after with PC:PS liposome but negligible'reversal upon pre-blocking with PC2Chol liposome ting the role of SA in the liposome responsible for PC:SA mediated ncer effect.

Example 4 Identification of the role of phosphatidylserine expressed on the surface of tumor cells in anti-tumor efficacy of PC:SA liposomes by ing the effect of PC:SA liposome on different cells after pre-blocking the cells with annexin V for 30 min DJ ‘4) To investigate the roleof PS in interaction with mes, PS was blocked by preincubation of Bl6FlO and K562 cell lines with annexin V prior to addition of PC:SA liposomes.

Cell suspensions in g buffer were incubated with or without annexi". ‘v’ at 37"C for 30 min. Cells were incubated with PC:SA liposomes (20-140 itle with respect to PC) for 2 h. Viability of the cells was assayed by MTT ion (22). Capability of PC:SA liposomes (I40 ug/ml)to induce 90% on K562 cell line and killing activity at 120 min, was drastically reduced to 50%, through annexin V blocking of surface PS of the cell line indicating significant PS-mediated killing. Non—specific negative—positive charge interaction with other membrane components may be responsible for the ing 50% killing activity (fig. 4A). Similar effect was observed in case ofBl6FlO cell line (fig. 48).

Example 5 Effect of camptothecin entrapped PC:SA'on different cell lines id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114"

[000114] Since PC:SA liposomes showed profound cytotoxicity on different cell lines, we proceeded on to entrap the anticancer drug camptothecin (CPT) in them and compared the cytotoxicity of CPT ped in PC:SA REV, DRV and MLV liposomes with equivalent doses of free drug. The molar ratio of the drug entrapped liposome was 7(PC): 2(SA): 0.7(CPT). We compared the cytotoxicity of CPT entrapped in PC:SA REV, DRV and MLV liposomes with equivalent doses of free drug on Bl6FlO cells. Figure SB revealed that a dose of 7ug/ml of free CPT showed 38% ity (fig. SB) whereas the same amount of drug entrapped in 140 ug/ml of PC: SA MLV liposome with respect to PC showed about 0% viability (fig. SA) after 2 h of treatment. Similarly‘2.3% and 1.3% cells were viable with I40 ug/ml of REV and DRV liposomes (drug entrapped) tively.(fig. SA) while with the lent amounts of free CPT (i.e. 3.9 ug/ml for REV and 6.] ug/ml for DRV) about 38% of cells survived'(fig. SB) after 2 h of treatment. Thus, drug ped in PC:SA liposomes showed more effective in killing the melanoma cells than corresponding amount of free drug. There was no significant difference in the g profile of CPT entrapped PC:SA REV, DRVgor MLV liposomes. r, PC:SA REV liposome showed slightly'lesser cytotoxicity at the lower doses. We wanted to determine the anti tumor y of CPT entrapped PC:SA liposome on EAC, and rat C6 glioma, human PBMC of y donor and SW480 cell lines after 2 h oftreatment. A dose of7 pg/ml of free camptothecin entrapped in I40 pg/ml of PC:SA showed effective killing effect on EAC, rat C6 glioma, SW480 and U937 cell lines and with l3% (fig. 5C), 2% (fig. 5E), 10% (fig. 5]) and 3% (fig. 5]) viability respectively but very less effect was ed on PBMC of healthy donor showing 71% viability at the same dose of liposomal CPT (fig. 50). A dose of7 pg/ml of free CPT showed 56% (fig. 5D), 71%' (fig. 5F) viability on EAC and C6 glioma cells respectively whereas it showed no killing effect on and normal human PBMC (fig. 5H). SW480 (fig. SJ) and U937 (fig. 5L) cell lines.

The results ed CPT entrapped in PC:SA liposomes was more effective in killing different cells than corresponding amount of free liposome and drug.

Example 6 Effect of doxorubicin entrapped PC:SA on different cell lines Next we compared the cytotoxicity of DOX entrapped in PC:SA MLV liposomes in the molar ratio 7(PC): 2(SA): 0.5(DOX') with equivalent doses of free drug on U937'and rat C6 glioma cell lines. Figs; 68, 6D and 6F revealed that a dose of 7pg/ml of free DOX showed 68%, 3% and 45% viability on U937, rat C6 glioma cell lines and EAC cells respectively whereas the same amount of drug entrapped in 'l40 pg/ml of PC:SA MLV liposome with respect to PC showed about 8%, 4.3% and l'8% viability on U937 (fig. 6A), rat C6 glioma cell lines (fig. 6C) and EAC cells (fig. 6E) respectively after 2 h oftreatment. Thus, DOX entrapped in PC:SA liposomes showed more effective killing effect on U937 and rat C6 glioma cell lines than corresponding amount of free drug.

The result indicates a synergistic anticancer efficacy of the PC:SA entrapped drug as compared to free drug or liposome. The synergistic effect ensUres low dosage of ent and decreased chemotherapeutic toxicity of the drug.

Example 7 Flow cytometric analysis of Pl-Annexin V to quantify tic effect of PC:SA liposome on different cell lines Detection between the intact viable cells, early apoptotic cells, and late _ tic or dead cells sis) can be carried out with Pl-annexin V double staining.- Thus, we performed this assay to explore cell apoptosis, Since the MLV form of PC:SA liposome showed m drug entrapment, nd killing , and was easy to prepare, we proceeded with it for the further experiments.

Untreated and liposome treated cell lines (5X10S cells/ml) were washed and resuspended in annexin V binding buffer (l0 mM HEPES, 150 mM NaCl and 2.5 mM Ca‘Clz) at .pH 7.3. In order to detect the translocation of phosphatidylserine from inner cell membrane to outer cell membrane (a characteristic feature of cells undergoing apoptosis), cells were subjected to flow cytometric analysis after staining with annexin V-FlTC and Pl. (FlTC annexin V Apoptosis Detection Kit ll BD PharmingenTM) (32).

For annexin V based apoptosis analysis , RAW264.7, human PBMC, K562, U937 and MOLT4 cells were analysed after treatment with the highest dose. (140 pg/ml) of PC:SA (MLV) liposome with respect to PC. The untreated control Bl6FlO, K562, U937 and MOLT4 human .PBMC cells showed about 79.4%, 94%, 97.1%, ' 94.5% and 95.2% viable cell population (FlTC— annexin V and Pl- negative) respectively. l8.4%, 4%, l.8%, 3.9% and 2.9% were PS sing cells (FlTC — annexin V positive and Pl negative) respectively (figs. 7Aa, 7Ba, 7Ca, 7Da, and 7Ea).

After treatment, however, a shift in the cell population towards early apoptosis was observed in which there is an increase in FlTC-annexin V positive and Pl negative cells.

. The percentage of viable cells for , K562 and U937 cells now deereased from 79.4%, 94% and 97.1% to about 33%, 37.3% and 66.7% respectively whereas the percentage of cells showing annexin V—FlTC positive and Pl negative increased from 18.4%, 4% and 1.8% to 66%, 60.3% and 30.8% respectively after ent for 2 h with 140 ug/ml of PC:SA liposome (figs. 7Ab, 7Bb, 7Cb). When K562 and MOLT4 cells Ln were treated with PC:SA liposome for 4 h with 140 ug/ml of PC:SA liposome the percentage of viable cells decreased from 94% and 94.5% to 20% and 50.93% respectively whereas the percentage of cells showing annexin V — FlTC positive and Pl negative increased to 74.2% and 40.87%. A certain percentage of MOLT4 cells ) and K562 cells (5.7%) also showed both annexin V-FlTC and P1 positive indicating late apoptosis (figs. 7Db, 7Bc). Thus, on treatment the PS content of the cells increases which indicates that apoptosis is being d by PC:SA MLV liposome but cell membrane ity is still maintained. Non~cancerous cells like normal human PBMC were also treated with the same dose and ons with MLV' PC:SA liposome. When normal human PBMC were treated with 140'ug/m1 of PC:SA liposome for 4 h, percentage of viable cells decreased from 95.2% to 86.7% whereas a small percentage of cells (9.6%) showing annexin V—FlTC positive and P1 negative (fig. 7Eb). The results revealed that 140 ug/ml PC:SA (MLV) liposome showed apoptotic mode of g activity on B16F10, K562, U937 after treatment for 2 h by binding with annexin V-FlTC where as K562 and MOLT4 cell line after treatment with 140 ug/ml of PC:SA (MLV) showed both early and late .mode of cell death. A very less percentage of PBMCs were found to be tic after treatment with 140 ug/ml of PC:SA liposome; Example 8 Flow cytometric analysis of ion in mitochondrial membrane potential (Aw) The mitochondrial membrane potential is a hallmark for apoptosis. Apoptosis is usually associated with rization of mitochondrial membrane potential (Aw). ln non apoptotic cells, .lC-l exists as a monomer in cytosol (green) and accumulates as aggregates in the mitochondria, which appear red. 1n apOptotic and necrotic cells, .lC-l exists in ric form and stains the cytosol green.

Cells (5X105/ml) were treated with PC:SA liposome or CPT entrapped in PC:SA liposome (140 ug/ml with reSpect to PC) for 2 h and centrifuged at 2000 rpm for 5 min.

Supernatant was carefully removed. 0.5 ml of freshly prepared 5,5l,6,6'—tetrachloro— l,l',3,Y-tetraethylbcnzimidazolocarbocyanine Iodide (JC-l) working solution containing » l:l00 JC-l stain was added to each . The pellets were ended. Cells were incubated in JC-l g sOlution for IO—IS min at 37 °C in a C02 incubator. Cells" were washed twice following incubation. Cells were then analyzed by flow cytometry.

(BDTM MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit) (33). To detect the s in A"), Bl6FlO cells were treated with MG ug/ml PC:SA MLV liposome, 7 ille of CPT entrapped in I40 pg/ml PC—SA MLV liposome and 7 ug/ml of CPT for two hours and analysed by flow cytometry. Fig. 7A shows typical FLal/FL-2 dot plots for JC-l staining of Bl6Fl0 cell line. Untreated Bl6Fl0 cells were Without sis, which have 96.8% red fluorescing J—aggregates and 3.1% green fluorescing monomers (fig. 8Aa). The increase in green fluorescing monomers 7%, 59% .15 and 73.5% shown in the lower part indicate apoptoti-c cells due to the treatment for 2 hours with 7 )4ng of free CPT (fig. 8Ab), 140 ug/ml PC:SA MLV liposome (fig. SAC) and 7 ug/ml of CPT entrapped in MO ug/ml PC:SA MLV liposome (fig. 8Ad) tively. To detect the changes in Aw, K562 cells were treated with I40 ug/ml of PC-SA MLV liposome for 2 hrs, and analysed by flow try. Fig. 14 shows typical FL-l/FL-2 dot plots for JC—l staining of K562 cell line. Untreated K562 cells were without apoptosis, which have 83.1% red fluorescing .l-aggregates and l5.6% green fiuorescing monomers (fig. 88a). The increase in green fluorescing monomers 47% shown in the lower part indicate apoptotic cells due to the treatment with MG ug/ml PC:SA MLV liposome for 2 hours (fig. 83b). The s revealed that untreated cells were without apoptosis, which have more red fluorescing .l-aggregates and less green fluorescing monomers. The increase in green cing monomers shown in the lower . part indicate apoptotic cells due to the treatment with PC:SA liposome and PC:SA-CPT respectively.

Example 9 DNA Cell cycle analysis Treatment of tumor cells with cytotoxic agents usually results in the breakdown of the cell cycle ‘machinery, the cells subsequently entering into programmed cell death or apoptosis. Cell growth arrest occurs in response to cell damage due to oxidative stress or DNA damaging chemicalsThese stresses induce the tumor suppressor protein p53, which arrests the cell cycle in G] or Gé/M. P53 is a transcription factor that represses expression of certain cyclins and Cdks, and directly induces the Cdk tor p21. Cell growth arrest allows cells to repair damages. Sub— '10 GO/G, accumulation is usually considered as an tic death profile. Cell cycle analysis was done to observe the effect of PC:SA me on the cell cycle ery ofthe different cells and also to see the mode of action of the liposome. The cationic lipOsome-caused anticancer effect by either apoptosis or via cell cycle arrest in different cell lines. This experiment has a far reaching implication as the mode of action can give us a clear idea ofthe cell cycle ns that are involved in the process and can be targeted for future al intervention.

Cell cycle analysis was performed. , untreated or PC:SA- treated cells were washed twice in PBS[ Pelleted cells were fixed in 70% cold ethanol and incubated overnight at 0C. After two washes in PBS,‘ the cells were resu5pended in 2O 0.5 ml of Pl (10 ug/ml in PBS) containing RNaseA (248 ‘U/ml), and the mixture was incubated for 20 min in the dark at room ature The fluorescence intensity of Pl was then analysed with a FACS Calibur flow cytometry and Cell Quest software (28).

Go/G. proportion in untreated Bl6F10, K562, U937, RAW 264.7 and PBMC, is 66.4%, 52.11%, 38.2%, 77.6% and 96.4% respectively. Sub Clo/G. represents 8%, 5.5%, 3.34%, 3.9% and 2.1% of cells respectively (figs. 9Aa, 9Ba, 9Ca, 9Ea and 9Fa).

Following 2 h of 140 pg/ml of PC:SA (M LV) liposome with respect to PC exposure on 816F10, K562 and U937 the (30/01 represents 53.5%, 30.8% and 30.6% of cells 'reSpectively while the sub GO/G; increased up to 31.9%, 37.69% and 6.13% respectively showing appearance of apoptotic nuclei (Sub GO/Gl peak in DNA cell cycle analysis) (figs. 9Ab, 98b and 9Cb). After 4 h treatment of U937 cells sub GO/G] further sed to 12.72% (fig. 9Cc). Gz/M represents 13.63% of cells in untreated U937 cell line (fig. 9Ca). Following 2 h and 4 h of 140 ug/ml of PC:SA (MLV) Lf‘l liposome expOSure. the Gz/iVi increases up to 14.41% and 15.85% respectively. (figs. 9Cb and 9Cc). ln untreated MOLT4 ce11 line, GO/G, proportion is 69.8% and G2/M represents 13.8% ofcells_(fig. 9Da). Following 4 h of 140 ug/ml of PC:SA liposome (MLV) exposure, the Go/Gl represents 62.2 % of cells while the Gg/M increases up to 21.3%. (fig. 9Db). Thus 140 ug/ml of PC:SA Iiposome exposure leads to Gz/M arrest in‘U937 and MOLT4 cell lines, When non—cancerouscells'RAW 264.7 and PBMC cells were treated with 140 ug/ml of PC:SA me(MLV) no significant increase in either sub 00/0} or (32M was ed. Only 4.7% of the RAW 264.7 cells were in sub Go/G. phase after treatment with 140 ug/ml of PC:SA Iiposome for 2 h (fig. 9Eb).

Similarly very negligible percentage of PBMC cells i.e. 2.1 % were in sub Go/Gl phase aftertreatment with 140 ug/ml of PC:SA Iiposome for 4 h (fig. 9Fb).

In the above cell cycle analysis it is evident that PC:SA at 140 ug/ml of treatment ed Gg/M phase in MOLT 4 in 4 h oftreatment. 1n U937 PC:SA at 140 ug/ml of treatment for 2 and 4' h arrested Gz/M phase as well as appearance of apoptotic nuclei as evident by sub Go/Gl peak. Whereas in K562 cells treatment of PC:SA causes apoptosis within 2 and 4 h of treatment as evident by sub GO/Gl peak.

PC:SAtreatment also causes appreciable apoptosis in 0 cells as evident by sub GO/G, peak. It showed only marginal effect on .7 and PBMC of healthy _ donor.

Example 10: Measurement of Reactive Oxygen Species (ROS) Level [0001211 ve oxygen species (ROS) are long known as general mediators of apoptosis. The dye H2DCFDA which is used for ROS estimation is freely permeable- across cell membranes and is incorporated into hydrophobic regions of the ce11. The e moiety is cleaved by cellular esterases, leaving impermeant. nonfiuorescent, 2',7’-dichlorodihydrofluorescein (H2DCF). The H2DCF is oxidized by reactive oxygen species to dichlorofluorescein (DCF), which emits fluorescence at 530 nm in response to 488 nm tion. * 2] K562, U937 and MOLT4 cell lines at 5X105 ml were treated with 40 S ug/ml and 140 ug/ml of doses ofPC:SA MLV liposome with respect to PC for 4 h and Bl6FlO and RAW 264.7 were treated with same doses for 2 h. Additionally K562 cells were treated with different trations of PC:SA Iiposome for 2 h. For each cell line an untreated control was kept. Cells were centrifuged at 4000 rpm for 3 min to remove the culture media and were washed twice in 0.02 M PBS by centrifugation at -10 4000 rpm for 3 min. The pellet was then suspended in 0.02 M PBS and was loaded with 10 uM Carboxy-DCFDA followed by incubation for 30 min at 3 1 °C in waterbath.

Carboxy-DCFDA is acell-permeable indicator for ROS that is non fluoreScent until the acetate groupsare removed by ellular esterases and oxidation occurs within the cell. When oxidized by various active oxygen species, it is irreversibly converted to the fluorescent form, DCF. Fluorescence was measured h a ofluorimeter (LS 3B;‘PerkinElmer, USA) using 499 nm as excitation and 520 nm as emission wavelengths. Data obtained as fluorescence intensity unit. The data were normalized to normal values, which was expressed as l00% (35). Human PBMC of healthy donor after treatment with 40 ug/ml and 140 ug/ml of PC:SA (MLV) liposome for 4 h ‘ ‘ 20 showed no increase in ROS level. U937, K562 and MOL'T4 cell lines showed increase in ROS level up to-200%, 300% and 200% respectively after treatment with 40 ug/ml of PC:SA (MLV) liposome for 4 h where as ROS level increased up to 400%, 405% and 300% respectively after treatment with I40 ug/ml of PC:SA (MLV) liposome for 4 h. No change in ROS level was observed in RAW 264.7 cell line treated with 40 ug/ml and MO ug/ml of PC:SA (MLV) Iiposome. ln Bl6F10~ cell line treatment with 40 ug/ml of PC:SA (MLV) me for 2 h showed an increase in ROS level up to 300% and treatment with I40 rig/ml of PC:SA (MLV) liposome for 2 h showed an increase in ROS level up to 500% (fig. l0A).

Different doses of PC:SA liposome (20-140. pg/ml) treatment for 2 h led to increase of intracellular ROS formation compared with untreated cells in K562 cell line (fig. lOB). The results revealed that K562, MOLT4, U937 and Bl6FlO, after treatment with PC-SA liposome led to se of intracellular ROS formation ed with control cells which was measured by conversion of HgDCFDA to 2,7— dichloroflurescein. RAW 264.7 and PBMCs after treatment with PC:SA did not show . increase in ROS.

Example 11:' ion of caSpase dependent and independent mode of cell death Treatment with caspase inhibitor Z—VAD-fmk reduced the ROS generation and also inhibited PC:SA me —induced apoptotic cell death, indicating the role of mitochondrial ROS in PC:SA—induced cell death. K562 cells were preincubated for 2 h with pancaspase inhibitOr Z~VAD-FMK (IO pM), washed in PBS, and subsequently incubated with different doses of PC:SA liposome (20—140 pg/ml) with respect to PC._ ity ofthe Was checked by MTT assay as described above (36). The results » revealed that Z—VAD-fmk treatment reversed the killing effect of PC-SA liposome on K562 cell as indicated by MTT assay. In Z-VAD—fmk treated cells the t of vialble cells increased from 4% to 29%, 37% to 68%, 49% to 77% and 57% to 75% at I40, I20, l00 and 80 pg/ml doses of PC:SA liposome respectively after 2 h of treatment. Thus indicating caspase dependent mode of cell death (fig. l 1).

Example 12: Detection of morphological s in U937 cells on treatment with . PC:SA liposome by Transmission Electron Microscopy (TEM) Surface logy of PC:SA liposome-treated cancer cells was d by ission electron microscopy (TEM). Briefly, cells were fixed in 3% glutaraldehyde in PBS, post-fixed with l% 0504 for 16—20 h, gradtially dehydrated in ethanol and finally embedded in SPURRT resin. Thin cut sections were stained with uranyl-acetate and lead acetate and were observed in a .lEOL-IOOCX electron microscope (34). Electron microscopic study of untreated and treated (40 pg/ml and 140 ug/ml of PC:SA liposome with respect to PC) U937 cells revealed a stark ence in the morphology of the cells. Fig. 12a shows that TEM representation of control untreated cells With round shape and intact membrane. Fig. l2b shows that treatment of U937cells with PC:SA liposome (40 ug/ml) causes the morphology ofthe cells to . Disruption of membrane integrity was seen. Fig. l2c shows that after treatment with the highest dose of PC:SA liposome (140 pg/ml), the membrane of some cells have egrated and large vacuoles are formed as well as depletion of electron-dense cytoplasmic material ting that the cell death is in process. Hence, TEM observations indicate that membrane disruption occurs on interaction of PC:SA with PS on the cell membrane ofcancer cells leading to killing ofthe cell.

Example 13: Study of mechanism of anti-cancer effect of PC:SA 6] Western blot analysis was'done-to ate the molecular mechanism of PC:SA-mediated effect. The experiment ates the different kinases that might be involved in. the mediated killing effect of cancer cells. The result of immunoblot analysis has a far-reaching impact as this shall clearly demonstrate the pathway involved in the process. tion. of ERK stimulates downstream ing cascades and also modifies transcription causing apoptotic changes. Caspase 9 when cleaved gets activated and forms part of the apoptosome complex and activates caspase 3 downstream which is also cleaved by PC:SA treatment. lnv‘olvement and activation of ERK indicates MAPK mediated apoptotic pathway. Involvement of p2l and and other cell cycle proteins'indicates the hindrance of cell cycle pathway as a probable mechanism for PC:SA mediated anticancer effect in some cell types. PC:SA mediated killing activates Bid which is an abundant pro-apoptoticprotein of Bel-2 family and is crucial for death receptor-mediated apoptosis. [000.127] The tphosphatidylinositolkinase (Pl3K)/serine/threonine kinase (Akt) ing pathway is essential to the survival and eration of human cells, and constitutive activation of this pathway is thought to play a critical role in the progression of human hematologic malignancies. Inhibitors of this pathway have been shown to induce apoptosis in isolated leukemia, lymphoma, and myeloma' cells. egulation of p-Pl3K indicates apoptotic mode of cell death in PC:SA treated cells.

Bl6FlO and RAW 264.7 cell lines were treated with,PC:SA MLV liposome £11 at Mt) pig/ml ‘with respect to PC for 2 h; U937 cells treated with graded ‘ were ‘ concentrations (20-l40 ug/ml) of PC:SA liposome with respect to PC for 2h. Untreated cell lines served as controls. The tive cells were kept in RlPA buffer and PMSF ght at -80°C. Next day the sion was centrifuged at 8000 rpm for 10 min and the supernatant containing the extracted proteins were collected. The protein contentof the extracts was estimated by Lowry's method. Equal amount of proteins (100 ug each) were taken and heated with OI volumes B-mercaptoethanol (4x loading buffer with loading dye) for 5—8 min at 80—90 °C and subjected to electrophoresis on % GE. The proteins were electrophoretically transferred on to nitrocellulose membranes. The membranes were blocked with 5% BSA and subsequently Washed-3 times with. TBST. The membranes were reacted with anti-ERK, anti-phospho—ERK, anti—Caspase 8, anti—cleaved Caspase 9, anti—p2l, anti-p38, anti—phospho—p38, anti—Bid, anti-pPl3K'and anti—B-Actin y antibodies at l/lOOO dilution each and kept at 4°C under constant shaking condition. overnight. Next day they were brought back to room temperature and then washed thrice with TBST. The blots were developed using respective HRP coupled secondary antibodies at ["000 dilution and kept at-~37 °C for 2 h. The blots were then thoroughly washed 4 times with TBST. Bands were visualized using chemiluminescent substrate, Luminol (Super signal West-Pico Chemilluminent Substrate). ln the chemiluminescence reaction adish peroxidase-catalyzes the oxidatiOn of luminol in presence of hydrogen peroxide into a t (3- aminophthalate) which emits light when it decays. This light was quickly ed in hardcopy on X-Ray films (37).

For estimation of protein by Lowry’s method: rd on (known conc : 20ug/20ul) was made by adding 20 ul BSA + 80 pl O.l N NaOH + 500 pl ne CuSO4 on + 50 pl Folin’s Ciocalteu reagent. Sample solution was made by adding l0 pl sample (whose protein content is to be measured) + 90 pl of 0.] NaOH+ 500 pl ne CuSO4 solution + 50 pl Folins Ciocalteu reagent. Blank (buffer for autozero) was made by adding l00 pl 0.1N NaOH + 500 pl alkaline CuSO4 solution + Folin’s Ciocalteu reagent. The samples were vortexed and kept in dark for min. Optical density (OD) was measured using a spectrophotometer (Pharma Spec UV-l700), at 750 nm. Protein content of the s were calculated with respect to standard (38).

Stripping for reprobing western blots: The stripping buffer (20 ml SDS 10%, 12.5 ml Tris HCl pH 6.8 0.5 M, 67.5 ml ultra pure water and 0.8 ml [5- mercaptoethanol) was warmed to 50°C in rbath. The blot was taken in a tight plastic-box and immersed in the stripping buffer and was incubate at 50 °C for up to 45 min with some ion. The buffer was then disposed off and the membrane-rinsed thoroughly under running tap water and then in TBST for several times as a interval of 5 mins until the B-mercaptoethanol smell is gone since traces of B—mercaptoethanol will damage the antibodies. The membrane is then ready for the blocking stage and proceeds as per rest of the western blotting procedure (Abcams protocol). lmmunoblot analysis trated that I40 pg/ml of PC:SA treatment with respect to PC for 2h does not change the expression of ERK (fig. l3Ab), or Caspase 8 (fig. 13Af) but causes an appreciable increase in the level of p21 (fig. l3Aa) phosphorylation‘of ERK (fig. l3Ac), cleaved caspase 9 (fig. l3Ad) and caspase 3 (fig. l3Ae) in Bl6FlO cells. RAW 264.7 was used as a control cell line. Beta actin was used as loading control (fig. l3Ag). The result clearly demonstrates the involvement of ERK activation in PC:SA-mediated anticancer effect. The increase in the level of cleaved e 9 and and caspasei3 is a. clear indication of caspase-mediated apoptotic pathway being involved in PC:SA mediated response. p2] involvement indicates the role of cell cycle machinery being invOlved in the PC:SA-mediated se. U937 cells treated with different trations (20-l40 pg/ml) of PC:SA liposome with respect to PC for 2h results in increased phosphorylation of ERK and activation of pro— apoptotic molecule called Bid (fig. ISB). The study in fig.‘ 14 was undertaken to ascertain that 140 pg/ml of PC:SA liposome down-regulates the PlBK/Akt signaling pathway in K562 cell line treated for 90 and 120 min concurrently with induction of apoptotic cell death.

Example 14' Effect of PC:SA liposome on Ehrlichs ascites carcinoma (EAC) [0001321 Based on the promising in—vitro s we have initiated studies looking into - the effect of PC:SA me iii-viva. Ehrlich Ascites Carcinoma (EAC) at 2X 1 06 cells was injected intra-peritoneally into 6 weeks old Swiss Albino mice. After 3 days of EAC injection, the mice were treated intraperitonealiy or intravenously with 1.7 g/kg body weight i.e 60 mg of PC:SA or PC:Chol liposomes/mice with respect to PC. The animals were observed for another two weeks and then sacrificed and photographed to study the effect of liposomal treatment. lntraperitoneal fluid was also taken out and the cell counts were done to determine the efficacy of PC:SA treatment (39).

The effect of PC:SA liposome on the body s ofthe EAC—bearing mice were examined. On day 14 the body weights of EAC-injected control mice was 39 gm and no significant difference was found in EAC-injected mice treated with PC:Chol liposome (39 gm). In st in jected mice treated with PC:SA liposome (i.v) a significant difference was found in the body weights (29 gm). The effect of PC:SA on the accumulatiOn tes fluid was examined on day 14. The s ofthe fluid in the jected control mice were very large with no difference in EAC-injected mice treated with PC:Chol liposome. in contrast in EAC-injected mice treated with PC:SA liposome the ascites fluid was reduced to one-fourth of the volume in EAC- injected controlgrouprhe effect of PC:SA me on the number of EAC cells in PC:SA treated and untreated carcinoma-bearing mice was also determined. The number of EAC cells in EAC-bearing control mice was very large. When EAC- injected mice were treated with PC1Chol liposome there was no change in the EAC cell number whereas ISAC—injected mice d with PC:SA liposome showed almost 1/200th of the cell number in control mice (fig; 15 and table 4). The results revealed thata single shot injection of 60 mg of PC:SA (i.p. and i.v.) inhibited the growth of U1 Ehrliclr ascites carcinoma in Swiss albino mice.

TREATMENT "’"E IGHT 0F NUMBER OF VOLUME 01" VOLUME 0F ANIIVIAL (gm) EAC CELLS PERITONEAL PACKED . FLUID (ml) CELLS (ml) , s N=3 (mo Din!) N'=t CONTROL 313 4:13 PC:SA-601mg :_ - 0.0710315 (1P) PC : SA-ISU mg 0 ,0: :01} 12 (LY) PCrfiflwlvffl' mg (IA-4') Table 4 Example 15 Effect of PC:SA and CPT-entrapped liposome on tumor development in syngenic black mice ctive aspectziAs PC:SA and CPT entrapped PC:SA liposomes inhibited cell proliferation in vitro, .we first examined its protective effects on tumor cell proliferatiOn in vivo by injecting 2Xl06 BI6FIO cells ated for 2 h with 7 mg/kg body Weight i.e 140 pg of PC:SA (respect to PC) alone, and 350 pg/kg body weight i.e 7 pg CPT entrapped in liposomes in C57BL6 mice subcutaneously. The mice were sacrificed after 2| days and tumor growths were observed. Tumor volume in Bl6Fl0 cells injected control mice was very large (7.8 cum) in comparison to mice pre—treated with 140 pg of PC:SA liposome (I56 cm3) with r reduction in tumor volume to almostnegligiblelevels (0.78 cm3)wvhen pre-treated with 7 pg CPT entrapped- liposome (fig. l6A and table 5); Therapeutic aspect: Next we examined the therapeutic effects of PC:SA and CPT ped in PC:SAi lviposomes on tumor cell proliferation in C57BL6 mice in vivo; Mice were injected with 2X l06 Bl6FlO cells on day 0 and then d on day 2 with MG ug'of-PC2iSA. liposome with. t to PC subcutaneously. in another set of experiment animals were injected with B l6Fl0 cells on day 0 and then treated on day 2 with" 7 ug' of CPT entrapped in I40 pg of PC:SA liposome. Untreated mice ed With Bl6FlO cells were kept as control. Mice Were sacrificed after 2l days and tumOr ‘ growths were observed. Tumor volume in control group was larger (3 l .46 cm?) than in PC:SA treated group (7.7 cm3) and in PC:SA-CPT treated group it was_ much smaller -(2.08 cm3) (fig: l 58' and table 6). We demonstrated that PC:SA liposome itself has anti- tumor activity 'by inhibiting cell proliferation in in vivo C57BL6 miCe and CPT- entrapped PC:SA'liposome was more effective than free liposome. 1 Table 5 Animals No of. animal Average body Tumor dimensions sacrificed wt111 gms Healthy 2 9 Height Length Blearlth control d ?9011M2 control PC:~SA 1.4cmiOJ treated CPT-V entrapped PC:SA treated.

Table ‘6 Example 16 Effect of PC:SA me on DEN induced hepatocarcinoma in Rats Adult male Swiss Albino rats, each weighing approximately IOU-IZOD g were divided into three groups ee animals each. Rats in group A were kept as normal ' y, these animals were injected with three doses of oliVe oil (0.5 ml) (i.p) at an interval of l5 days. All rats in the experimental groups were injected with three doses of DEN (i.p) 200 mg/kg body wt in 0.5 ml olive oil at l5 days interval. Group B animals were kept as DEN administered-control. Animals in group C were treated with three doses of 800 mg/kg body weight i.e 80 mg (in 0.8 ml) of PC:SA (i.v) from the 7th day of 1S1 DEN administration at l5 days of interval. At the end of 18 week starting from the lst day of DEN administration, the final body weight was measured and blood was collected from heart in the rats of each group. Serum aspartate transaminase (AST), alkaline phosphatase (AP) and serum alanine minases (ALT) were determined using a standard kit manufactured by Span Diagnostics Ltd.

At‘ter collection ot‘blood, all rats were dissected and their livers were isolated promptly and washed with cold physiological saline. Final liver weights of all animals were recorded and relative liver weights (RLW) were calculated, A part of the organ was fixed, in 10% formaldehyde and processed overnight, and paraffin wax embedded.

Sections were stained with hematoxylin and eosin (H&E) for histopathological. rGroup Relative liver "/0 se of L'Ver We'gh‘. . weight (RLW) RLW p (3;) Normal 6.67:I: 0.77 2.56:I: 0.10 DEN 7.67:0.61 3.3%: 0.17 lDEN +PC:SA . .99:b 0.17 3.07i 0.19 Table 7 id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136" id="p-136"

[000136] DEN (3 doses of i.p. 200 mg/kg b.wt at l5'day interval) treatment causes an increase in relative liver weight (RLW) in rats. PC:SA (3 doses of i.v. injection of 800 mg/kg body weight ie 80 mg in 0.8 ml at IS days interval) liposome prevented increase in RLW of liver in rats significantly in comparison to DEN administered . control group (table 7). 7} oxylin-Eosin-stained liver sections of normal rat showed hepatocytes are arranged in cords around hepatic vein forming hepatic s. Portal tracts are . DEN injected animals show dialated hepatic veins and hepatic micronodules separated'by thin fibrous septum. However PC:SA liposome have prevented the liver from developing hepatocarcinoma. Normal looking hepatic vein, portal tracts and hepatocytes are present here but some amount of rtal fibrOsis are not prevented (fig. 17). in the assessment of liver damage by DEN. the determination of enzyme levels was largely used. Serum SGPT, SGOT and ALP areithe most ive markers employed in the diagnosis of hepatic damage because these are cytoplasmic in l0Cation and are released in to the circulation of after cellular damage. In this study, an increase in the ties of'SGPT, SGOT and'ALP in serum evidenced the DEN—induced hepatocellular damage. The reduction of DEN-induced elevated plasma activities of these enzyme levels in animals treated with PC:SA liposome showed their ability to e the normal functional status of the damaged liver. The results of this study' clearly demonstrated that ‘PCZSA liposome exhibited potent hepatoprotective activity against DEN-induced hepatic damage ‘n rats (figs. i8A, ISB and 18C).

Example 17 Comparison of in vitro ECSQ of PC:SA and anticancer drugs on different cell lines 5X105 cells/ml of murine melanoma cell line Bl6FlO, rat brain astrocytes; ctal adenocarcinoma SW480 cell line, human colon carcinoma HCTl l6 cell line, rat brain astrocytes, human neuroblastoma SHSYSY, human hepatocarcinoma HepGZ, humancervical carcinoma HeLa, human breast cancer MCF7, adeno gastric carcinoma AGS, colorectal adenocarcinoma SW480 cell line human nic kidney HEK, ‘ mouse fibroblast N-lH3T3 and rat C6 glioma cell lines were seeded in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal .bovine serum, sodium pyruvatez 2 mM L—glutamine, penicillin, and streptomycin in 96 well- cell culture plate. 5X105 ml of murine hage cell line RAW 264.7, MOLT-4, MOLT3 cell line ed from human acute lymphoblastic leukemia), human ia cell lines K—562 and human lymphoma cell line U-937, healthy human PBMC, Ehrlichs ascites carcinoma (EAC') Were seeded in RPMI 1640 medium mented with l0°/o fetal bovine serum, sodium pyruvate, 2 mM amine, penicillin, and streptomycin in 96 well tissue culture plate. The cells were treated with PC:SA liposome (20—350 ug/ml with respect to PC) or free DOX (5004000 lug/ml) and free CAMPTOSAR (irinotecan hydrochloride, Which is a semisynthetic derivative of camptothecin) (500- l0,000 gig/ml). After 2 h oftreatment effects on cell viability were determined by MTT assay. EC50 values were determined by nonlinearregression analysis of concentration- response data.

PC-SA liposome selectively killed murine melanoma cell line Bl6Fl0: human neuroblastoma'cell line SH-SYSY: ctal adenocarcinoma SW480 cell line= human colon carcinoma HCTl 16 cell line, rat C6 glioma, MOLT—3, MOLT4 cell line (derived from human acute lymphoblastic leukemia), human leukemia cell lines K—562, human lymphoma cell line-U—937, PBMC of acute promyelocytic leukemia and AML patients, Ehrlichs ascites carcinoma cells (EAC) and adeno c oma AGS cell line U1 (half—maximal effective concentration (ECSO) = 65-170 rig/ml for 2 h ent, respectively) (Fig. 19a). r it showed weaker killing activity against non- cancerous and cancer cell lines including human embryonic kidney HEK, mouse fibroblast NIH3T3, macrophage cell line RAW 264.7, human peripheral blood mononuclear cells (PBMC), human breast cancer MCF7, human liver cancer HepGZ, human cervical carcinoma HeLa, and rat brain astrocytes (EC50=218-480 ug/ml), with less surface exposed PS (Fig. 19 a) indicating that PC-SA liposomes ly do not kill cells h an off-target mechanism.

Free CAMPTOSAR (irinotecan hydrochloride, which is a semisynthetic derivative of camptothecin) and doxorubicin (DOX) were tested for the s of these anticancer drugs on seven cancer cell lines Bl6FlO, K562, U937, rat C6 glioma, U937, EAC, MOLT4 and SW480 cell lines. Fig. 19 b revealed that EC50 values of free irinotecan hydrochloride (a semisynthetic derivative of CPT) were in the range of 800-5,000 itg/ml and the ECSO values of free DOX was in the range of 500 pg/ml — 800 rig/ml.

The above example revealed that free liposome PC-SA is itselfa very potent anticancer agent. In vitro studies shows that EC50 value of free liposome against some of the cancer cell lines is in the range of 65—l70 pg/ml whereas the EC50 value of free anticancer drugs like doxorubicin and ecan hydrochloride (a semisynthetic derivative of camptothecin i.e; its injection formulation) is in the range 'of 500-5000 rig/ml i.e. almost 8 to 30 times more than that of free liposome when treated for 2h. The free e is much more potent than free known anti-cancer drugs. The ECso value Of free liposome is higher t non-cancer and some of‘other cancer cell lines which have less PS content. Therefore, PC:SA lipsome is effective against PS containing cancer cells only and not against non-cancerous and less PS containing cancer cells.

Figure: 19: Comparison of the effect of PC:SA liposome and free anticancer drugs on cancer cell lines in vitro. The viability of all the cell types was measured by inhibition of 3-(4, S-dimethylthiozolyl)-2, 5-diphenyltetrazolium bromide (MTT) reduction to insoluble formazan by mitochondrial dehydrogenase after 2 h of treatment.

L11 The EC50 values of free PC:SA liposome (a), free irinotecan hydrochloride (semisynthetic derivative of CPT ) and DOX (b) for in vitro killing of cell lines. Error bars denote standard deviation of3 experiments.

The free liposome PC-SA is itselfa very potent anticancer agent. In vitro studies shows that EC50 value of free liposome against some of the cancer cell lines is in the range of 60-80 ug/ml s the EC50 value of free anticancer drugs like doxorubicin and irinotecan hydrochloride. (a semisynthetic derivative of camptothecin i.e. its injection formulation) is in the range of 700-5000 ug/ml i.e. almost l0 to 60 times more than that of free liposome when treated'for 2h. The free lipsome is much more potent than free known ancer drugs. The ECSO value of free liposome is higher against ncer and some of other cancer cell lines which have less PS content (Figure 19). Therefore, PC-SA lipsome is effective against PS containing cancer cells only and not against non- cancerous and less PS ning cancer cells.

ADVANTAGES OF THE PRESENT INVENTION l. PC:SA liposome in its 7:2 molar ratio is non toxic to normal human cells like PBMC, whereas some cancer cells are susceptible to the liposome. 2. The unique mode of selectivity towards PS, of various cancer cells and cmia tes indicates that the SA—bearing liposomes might be valuable as delivery system as well as therapy not only against cancers and leishmaniasis but possibly also against other diseases which have elevated e levels of negatively charged phospholipid, i.e. PS.

LIJ PC:SA liposome is ive in inducing apoptosis of various cancer cell lines without g‘ appreciable effects on normal human peripheral blood mononuclear cells and this might be valuable as therapy as well as for delivery against cancer cells with apoptotic defects; PC:SA. liposome. mediates apoptosis 0f various cancer cell lines by, mitochondrial membrane depolarization, generation of ROS, activation of caSpases, RK and dowrireguiation ofPl3K ed pathway.

PC:SA liposome is an alternate drug delivery system that is being used to enhance the therapeutic efficacy and reduce the toxicity of anticancer agents like camptothecin and doxorubicin. PC:SA liposome entrapped camptothecin and doxorubicin are liposomal formulations of camptothecin and doxorubicin ' ed to increase efficacy, safety and tolerability while potentially delivering higher doses tothecin.

SA-bearing liposomal anticancer drugs will help in the treatment of cancer with minimal detrimental side-effects.

It will provide a strong rationale for the use of our mal cytotoxic therapeutic agents for the treatment of human cancer. 8. It will be ofgreat importance to treatments in which toxic substances are needed to combat disease.

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Claims (16)

WE CLAIM :

1. Use of a liposomal formulation for the manufacture of a medicament for the treatment of cancer, wherein said liposomal formulation comprises phosphatidylcholine (PC) and stearylamine (SA) in a molar ratio of 7:2.

2. The use according to claim 1, wherein EC50 value of said liposomal formulation t a cancer cell line is in the range of 60-80 µg/ml.

3. The use ing to claim 1, wherein the medicament is formulated for administration at 3 doses of 800 mg/kg body weight with respect to PC against DEN induced hepatocarcinoma in a rat.

4. The use according to claim 3, wherein the ment is formulated for enous administration.

5. The use ing to claim 1, wherein medicament is formulated for stration as a single shot injection of 1.7 g/kg body weight with respect to PC in Swiss albino mice to inhibit growth of Ehrlichs ascites carcinoma (EAC).

6. The use ing to claim 5, wherein the medicament is formulated for intravenous or intra-peritoneal administration.

7. The use according to claim 1, wherein the medicament is ated for administration at a single dose of 7 mg/kg body weight with respect to PC against B16F10 melanoma in vivo.

8. The use according to claim 7, wherein the medicament is formulated for subcutaneous administration.

9. The use of claim 1, wherein said formulation is a synergistic liposomal formulation, wherein said synergistic liposomal formulation comprises phosphatidylcholine (PC), stearylamine (SA), and at least one anticancer drug selected from the group consisting of camptothecin (CPT) and doxorubicin (DOX), said formulation comprising atidylchloine (PC) and stearylamine (SA) in a molar ratio of 7:2.

10. The use of claim 9, wherein said formulation comprises phosphatidylchloine (PC), stearylamine (SA) and camptothecin (CPT) in a molar ratio of 7:2:0.7.

11. The use of claim 9, wherein said formulation comprises phosphatidylchloine (PC), stearylamine (SA) and doxorubicin (DOX) in a molar ratio of 7:2:0.5. (26575770_1):RTK 60

12. The use according to claim 9, wherein said synergistic liposomal formulation is prepared in a form selected from the group consisting of ation-rehydration vesicles (DRV), reverse phase evaporation e (REV), and multilamellar vesicles (MLV).

13. The use according to claim 9, wherein said cancer is selected from the group consisting of murine melanoma, rat glioma, colorectal adenocarcinoma, human colon carcinoma, chronic myelogenous leukemia, acute lymphoblastic leukemia and ascites carcinoma, and said treatment is an in vitro treatment.

14. The use according to claim 9, n the medicament is formulated for administration of a dose of said synergistic liposomal formulation of 20-140 µg/mL with respect to PC.

15. The use according to claim 9, wherein the synergistic liposomal formulation ses CPT, and the medicament is formulated for stration of CPT at a single dose of 350 µg/kg body weight entrapped in 7 mg/kg body weight of the said formulation with respect to PC for increased anti-tumor effect of the free liposome against B16F10 ma in vivo.

16. The use according to claim 15, wherein the medicament is formulated for subcutaneous administration. Council of Scientific & Industrial Research By the Attorneys for the Applicant N & FERGUSON Per: (26575770_1):RTK 61